Lab Notebook

Aim To train ourselves for protein purification. Indeed, upon seeing our design, the protein facility offered us the opportunity to train in protein purification, so that we would be ready for the purification of our designed proteins. For this training, we purified BirA proteins that were already induced with IPTG in E.Coli (strain K12). We did an affinity purification with Ni-NTA beads since the BirA contained a His tag, as well as our engineered proteins.

Protocol Protein purification PTPSP and SDS-PAGE

Remark

• We will run all the fractions on a gel, so solutions from every step. This includes the lysate, supernatant, all the washes and all the elutions. This is because until we see the band on a gel we have no idea where the protein is. From the results we will decide which fractions to pool (combine) and add protease (if tag removal is required).

• The first 4 elution samples were dialyzed overnight in 300 mM NaCl and 20mM HEPES 7.5

Results

We obtained a concentration c = 2.11 g/L, and a final protein volume of 17 mL

Analysis By using the Beer-Lambert law, we obtained a final protein concentration of 2.11 g/L after the purification and the dialysis. This is a pretty high concentration. By analysing the results of the SDS-PAGE we can clearly see a band around 30kDa in almost all the samples which confirms the presence of the protein. Since the protein appears to be produced in large quantities by E.Coli, we even find them in the two last washes. Then, as expected, we obtained the biggest amount of proteins in the first 4 elution samples, that is why we selected them for the dialysis. The last two elution samples contained the least amount of protein, which was expected as well.

Conclusion Overall, we successfully purified the BirA protein. This was a good training of the protocole we are going to use for the protein purification of our SR proteins. For the silk protein, we should change some steps of this purification procedure because of the solubility tag and of the CBD tag. Indeed, the latest may be a problem for the dialysis step, since we put the proteins in a cellulose membrane. Therefore we will research another method to avoid the cellulose back step.

Aim To express the 01a (mSA-silk-CBD) construct in BL21(DE3) and then purify the obtained protein.

Aim To do the transformation of BL21(DE3) cells with the 01a plasmid (mSA-silk-CBD). We used water as negative control to model the absence of plasmid. We used a GFP plasmid from the summer school as a positive control. Since the bacteria will be treated with Kanamycin, we expect to observe grown colonies from the 01a transformed plate and the positive control since the plasmid contains a gene resistant to Kanamycin, and the absence of colonies with the negative control, since there is no antibiotic resistance.

Protocol Bacterial transformation

Results

Analysis The negative control worked as expected (A). No plasmid were uptaken by bacteria, thus no resistance was obtained. The positive control worked also as expected (B). One can see a lot of colonies on the ampicillin plate, which proves that the bacteria transformation is successful in this case. Finally, the 01a plasmid transformation worked as well (C). We can observe some colonies on the Kanamycin plate.

Aim To amplify the plasmid 01a by picking a bacterial colony after bacterial transformation.

Protocol Colony picking

Remarks We put 6 mL of LB-Kana medium in two 15 mL falcon tubes. We did two colony pickings so that we have two liquid cultures for further experiments. We picked from two different colonies on the same plate.

Aim To grow the bacteria transformed with the plasmid 01a in a LB medium with Kanamycin to prepare big cultures and then to induce the transcription of the plasmid 01a containing silk fused to mSA, CBD and His-tag for protein expression with IPTG.

Protocol IPTG induction

Remarks We used 2 samples for this experiment. We picked two different colonies from the culture plate. Sample 1 comes from a colony picked by Charlotte the 09/08 and Sample 2 comes from a colony picked by Elodie the 09/08.

Results Our first OD₆₀₀ measurements results were too high (above the 0.4-0.6 range), so we had to do a dilution for each of the samples.

For the first OD₆₀₀ measurement, we didn’t resuspent the bacteria. We did the dilution we thought was sufficient (2:5 for sample 1 and 1:2 for sample 2), but by re-measuring the OD₆₀₀ we got too high values (0.746 for sample 1 and 0.953 for sample 2). Then we decided to do a second dilution on top of the first one (2:3 for sample 1 and 1:2 for sample 2) and this time we obtained OD₆₀₀ in the good range (see table 4).

Analysis Since the cell culture should have a final OD₆₀₀ < 6.0 for efficient processing, and that we obtained < 6 we can use our samples for the purification.

Aim To purify our induced silk fused to mSA, CBD and His-tag protein. To do so, we will use the MagneHis Protein purification kit from Promega and will follow their Magnetic Histidine Purification Protocol. The kit uses paramagnetic precharged nickel particles to link in a matter of minutes His tagged proteins from the bacterial lysate. Unbound proteins will be washed away with a specific washing buffer, and we will purify our protein of interest with specific elution buffers that contain imidazole.

Protocol MagneHis Purification

Remark

• We did not wait a long time for the elution step.

• We did two elution steps in total for each sample.

• When the samples were incubating at RT with the beads and the buffers, it began to form a heterogeneous mix with the beads at the bottom of the tubes and the supernatant at the top.

Results

Analysis Since the cell culture should have a final OD₆₀₀ < 6.0 for efficient processing, and that we obtained < 6, we can use our samples for the purification.

Aim To send the transformed bacteria with plasmid 01a from our overnight culture for sequencing to confirm that plasmid 01a was taken up by bacteria and that there is no disturbing mutation within the DNA sequence.

Protocol E.coli Night Seq

Results Sequencing alignment file

The global view of the CD’s culture sequencing results shows that the T7 forward primer matches in a part of the silk protein sequence and it distorts the sequencing. For the T7 Term primer, everything seems fine.

The global view of EM’s culture shows that the sequencing results can be analyzed further since the T7 forward and Term primers are matching the sequence.

The sequencing was obtained using Microsynth’s E.coli NightSeq. The open reading frame is intact and all of our inserts are in frame and present. However, we only managed to get small alignments with the sequence which is not as expected.

Analysis As one can see on Figure 3, the CD’s sequencing results are not as usual and cannot be analyzed further through a map or an analysis of the alignment with the sequence.

On the contrary, Figure 4 shows that the EM’s sequencing results can be analyzed further.

Globally, the reading frame is conserved and we don’t observe serious mutations in the sequence alignments (see Figure 5). However, we can notice some “N” mutations in the mSA sequence and a deletion of two nucleic acids at the end of the His-Tag after the STOP codon that damages the PspXI restriction site. Also, the sequencing stops at the beginning of the silk coding sequence, which might be normal since the silk has a lot of repetitive motives in its sequence.

Aim To purify plasmid DNA from our overnight culture of bacteria transformed with plasmid 01a, then send it to the sequencing center, to confirm that plasmid 01a was taken up by bacteria and that there is no mutation within the DNA sequence.

Protocol MiniPrep

Remarks

• We applied the protocol of 3ml of bacterial culture as starting material and used tubes of 1.5ml.

• We added 2 minutes to the centrifugation step that normally lasts 3 minutes because of last time.

• We did twice the washing steps with ERB + centrifugation and Column wash + centrifugation.

• Elution conditions : 40μl (that was a bad idea) with elution buffer

• For the NanoDrop, we pipetted 1.5μl on the machine.

• For the sequencing, we pipetted 20μl of our samples, for sequencing with primers T7 and T7 Term from the Standard Primer List of Microsynth.

Results

Analysis NanoDrop concentrations measured with the NanoDrop are very low and might show a lack of DNA in our MiniPrep samples. We thought at first that our bacteria might have died. But this is not the case since we did a big culture after and the corresponding OD₆₀₀ measurements showed that our bacteria proliferated. Another reason for the low concentrations might be that we eluted with more elution buffer than in the protocol (40μl). However, we are very uncertain this might be the reason why we have such low concentrations because it seems to us that this change would not have such a big impact on our MiniPrep samples. Otherwise, we don’t have any more clue about how to explain the low concentrations.

Sequencing As one can see on Figures 7-9 above, our sequencing results cannot be analyzed since the alignment for each sequence is too small and there is a lot of noise that makes the detection of bases difficult for Sanger sequencing. SnapGene refuses to even open the files because the quality of the sequencing is too low.

Conclusion We will do a Restriction-Digestion of our plasmids and run them on an agarose gel to identify if we have the good sequences and the good restriction sites. Danica will write to the company that synthetized our plasmid to ask them if they have any quality control of our plasmids. We will do another MiniPrep next week to see if we have better results (+ we need it for the Restriction-Digestion we aim to do).

Aim To run the SDS-PAGE of our samples from the mSA-N[AS]4C-CBD (01a with silk) protein purification, to find in which tube we can find most of our protein of interest. By the end of this experiment, we should choose the tube with the higher amount of protein, measure the purity and concentration of the protein and use it for the coating of the aerogel.

Protocol SDS-PAGE

Remarks We ran 3 gels in total: 2 identical 15-well gels with samples 01a-1 and 01a-2 (one for protein staining and one for Western Blot), and one 10-well gel with sample 01a-3 (control with no IPTG induction) for protein staining. For the 3 gels we loaded ~30 μl in the wells.

Results

Analysis For both samples, we observe exactly the same results. Moreover, no real difference in the strength of the signal can be noticed between the two different samples, which means that induction with 1mM or 2mM of IPTG doesn’t really change the result.

With our plasmid backbone we expect a molecular weight of 74.2 kDa for our silk fusion protein. As one can notice on Figure 10, no band at 74.2 kDa is noticeable on the gel.

Also, we are not sure that the plasmid backbone is as we wanted because of some problems in the sequencing we are facing. But we are optimistic regarding this matter since we can observe a band at approximately 74.2 kDa on the Western Blot (which was done afterwards).

In addition to these results, we obtain for all samples (1, 2 and 3) a lane around 45 kDa, which is not the expected size. This means that we have another protein that has been purified with our kit and that contains a His-tag or a hQ tag. Moreover, some contamination can be observed in most of the samples, especially in Elution 1 and around 17kDa. Finally, we cannot observe our protein of interest with this experiment.

Conclusion We cannot see our protein of interest in this experiment. To verify the presence of the protein, we will do a Western Blot which is more sensitive than the SDS Page. Indeed, by using conjugated anti-His antibodies, we should be able to detect each protein that contains a His-tag. A consistent contaminant was also seen in the elution lanes around 17 kDa. In this General consideration of the protocol, it is written that lysozyme used to lyse the cells will elute with the fusion protein and will produce a 12.5 kDa band on a SDS polyacrylamide gel. For future experiments, to prevent the binding of lysozyme to the MagneHis during the purification, we should include NaCl in the Binding/Wash Buffer. Finally, we should do some troubleshooting experiments to better understand which protein produces a band at approximately 45 kDa and where it comes from.

Aim To do a Western Blot of the gel from the sample 1 and 2 of the 01a construct (silk protein with 4 As module). We will use His-tag antibodies that bind the same His tag we used for purification in order to identify specifically the purified silk protein and see in which sample it is the most present.

Protocol Western Blot

Remarks We used gel with the silk protein (01a) from sample 1 and sample 2

Results

Analysis We observe similar results for both samples. However, with sample 2 we observe a band around 72kDa. Since we expect our silk protein to weigh around 70kDa, this last band must be our protein of interest. Thus, in this experiment it is better to use a 2mM IPTG for induction since it reveals best our protein on the Western Blot.

For both samples, we observe two unexpected lanes in the lysate. First a band around 130kDa and then a second one around 20kDa. This means that we have two His or hQ tagged proteins in the lysate in addition to our silk protein.

Unfortunately, we cannot see any signal from the elution sample, which means that we didn’t successfully purify our silk protein, which seems to have no affinity for our magnetic stand.

Conclusion In this experiment, we noticed that we haven’t successfully purified the silk protein since it is present only in the lysate. Moreover, we need to understand why we observe two other bands in the lysate, and where do these proteins come from. Since we didn’t add any protease inhibitors, we will try to use them when redoing the experiment to see if the lower band remains.

Aim To transform competent cells with our construct, express the corresponding protein and purify it. Overall, here we want to retrieve our induced Silk fused to mSA, CBD and His tag protein, to then use as a hydrophobic coating on the aerogel. To do so, we went to the protein facility to test a new purification protocol and new bacterial strains.

Aim To do the transformation of E.Coli Competent Cells using two different strains, BL21 and Rosetta, with the 01.a plasmid (Silk + CBD). We will plate the cells on agar plate containing Kanamycin, and we expect the cells that have integrated our plasmid to resist those antibiotics.

Protocol Bacterial transformation

Results After the overnight incubation, we observed the presence of colonies in both the Rosetta and the BL21(DE3) transformed cells plates but there were more colonies on the BL21(DE3) plate. The negative control plate did not show any colony, and the positive control plate was full of colonies, as expected.

After the overnight incubation, we observed the presence of colonies in both the Rosetta and the BL21(DE3) transformed cells plates but there were more colonies on the BL21(DE3) plate. The negative control plate did not show any colony, and the positive control plate was full of colonies, as expected.

Analysis The 01a plasmid transformation worked well : we can observe some colonies on both the Kanamycin plates (the one containing the BL21 transformed cells and the Rosetta transformed cells).

Aim To grow the two bacterial strains transformed with the plasmid 01a in a TB medium with Kanamycin to prepare big cultures and then to induce the transcription with IPTG of the plasmid 01a containing silk fused to mSA, CBD and His tag.

Protocol Colony picking

Remarks We used 2 samples for this experiment since colonies grew from the two bacterial strains: rosetta and BL21.

Results

Before the IPTG induction we obtained OD₆₀₀ that were between 0.6 and 0.8, which is the required range at PTSP to pursue with the induction. Then, we measured the OD₆₀₀ after the IPTG induction, and observed small values to the ones PTSP usually gets after induction (between 2 and 10).

Analysis In the gel we ran to see if there is the expression of our protein, we couldn’t see the expression of the silk protein from the 01a plasmid in any of the samples. However, since we obtained smaller OD₆₀₀ values than the ones PTSP is used to obtain after the IPTG induction (OD₆₀₀ between 2 and 10), we decided to do the purification from those samples. Indeed, it can be that the silk protein is present but in very small amounts and concentration. To check this assumption we should do the purification and run an SDS-PAGE gel as well as a Western Blot, and see if anti-His antibodies recognize the protein of interest.

Aim To purify our induced Silk fused to mSA, CBD and His tag protein, from both initiale samples coming from BL21 and Rosetta. To do so, we will use the Ni-NTA affinity beads and columns of the Protein Facility (PTSP). This purification method is used to purify His-tagged proteins. Unbound proteins will be washed away with a specific washing buffer, and we will purify our protein of interest with specific elution buffers that contain increasing concentration of imidazole. We make all the buffers ourselves.

Protocol Protein purification PTPSP

Remark

• Since our control (GFP fused with CBD and mSA) seemed to be stuck on the beads even after the second elution, we increased the imidazole concentration and did other elutions until reaching the stock concentration of 5M.

• We need to be careful now, because imidazole can be aggressive for the stability of proteins, and may enhance their denaturation. We thus need to find solutions to avoid this required increasing amount of imidazole in the elutions samples.

• We stopped at the first point of step 6 since we realized that the filter to concentrate the proteins as well as the dialysis membrane were made with cellulose.

Results We calculated the amount of the protein after purification by measuring the concentration to the nanodrop:

Storage We wanted to concentrate them, so we used a filter of 30kDa. However, we noticed later that this filter was made of cellulose. It is then possible that some protein has bound to the membrane. For this reason we decided to not do a dialysis of this protein to remove the excess of imidazole. Indeed, the dialysis membranes are mostly made of cellulose. We then Flash freeze in aliquot of 1.5 mL the proteins, to let us time to find a solution to remove the imidazole that can be a problem for the stability of the silk.

Aim To run the SDS-PAGE of our samples from the silk protein purification of both samples coming from BL21 and Rosetta, to find in which tube we can find most of our protein of interest. By the end of this experiment, we should choose the tube with the higher amount of protein, measure the purity and concentration of the protein and use it for the coating of the aerogel.

Protocol SDS-PAGE

Remarks

• We did two gels because we had done two purifications : one for the 01a in BL21 and one for 01a in Rosetta.

• We have two different supernatant samples because we did the sonication step of the purification twice since the control had a very green pellet even after the first lysis. This meant that not all cells were lysed during the first sonication, so we did it a second time to recover more proteins.

• We have no elution 3 (E3) here because we first increased the concentration to elute the control, and saw that the elution 3 wasn’t working so well to remove the green color of the beads. Thus we decided to not do this step in the other samples.

Results

Analysis In both the gels, we can clearly see a band around 100kDa, which is higher than the expected 74kDa. It can be that the protein didn’t denatured well, or because of its repeating sequence it kind of folded a bit which didn’t allow it to run so far. In any case this 100 kDa band means that we successfully purified a protein with a His-tag in both the samples. To check either this protein is the silk protein we are expecting, we will run the elution samples in a Western Blot.

Aim To do a Western Blot with the elution samples to see whether the protein we see around 100kDa is our silk protein that we hope we successfully purified. Since in theory we should have our protein of interest in the elutions samples, we mixed the elutions samples between rosetta and BL21. Which means that we fused both the E1, both the E2, etc… That is why, we will run in the western blot the pellet of 01a BL21, the supernatant 2 of 01a BL21, and the mixed elutions: E1, E2, E4 and E5.

Protocol Western Blot

Results

Analysis We clearly see a band around 100kDa. This means that a protein has been recognized by the anti-His antibodies. It allows us to confirm that this protein is indeed our silk protein that is a bit bigger than expected (74kDa). Moreover, we obtained a really good amount of it that we decided to flash freeze and store at -20°c to let us time to figure out how to remove the excess of imidazole.

We successfully purified the silk protein which is mostly present in elution 1 and 4 in good amounts for a 300mL culture

Aim To transform competent cells with our new construct, express the corresponding protein and purify it. Overall, here we want to retrieve our 01a (mSA-silk-CBD) construct that does not contain the added site of Gen Script. To do so, we went to the protein facility to redo the purification protocol from 24.08.2022.

Protocol Bacterial transformation

Remarks

• Here we used 25 μl of E.Coli competent cells and 2.5 μl of the new 01a plasmid (mSA-silk-CBD).

• We didn’t have gas anymore so we couldn’t work with the flame, therefore we worked under the laminar flow hood for sterile techniques.

• We didn’t do any controls because we didn’t have enough plates and we already did many transformation controls.

• We used 200 μl of SOC medium instead of 100 μl since we started with more cells.

Results

Analysis The 01a plasmid transformation of BL21 competent cells worked well : we can observe some colonies on the Kanamycin plates. We can continue the process by doing colony picking in a sterile environment.

Aim The aim of this experiment is to amplify the 01a (mSA-silk-CBD) plasmids by picking bacterial colonies from bacterial transformation made on 2022.09.13 and to grow bacteria in bigger liquid cultures.

Protocol Colony picking

Remarks

• We put 10 mL of LB-Kana medium in a 15 mL falcon tube.

• We incubated the cultures for about 20h.

Aim The aim of this experiment was to grow the BL21 competent cells transformed with the new 01a (mSA-silk-CBD) plasmid in a TB medium with Kanamycin to prepare big cultures and then to induce the transcription with IPTG of the 01a plasmid.

Protocol IPTG induction

Remarks We directly did 1.3L of big cultures. So we put 1.3L of TB+Kana and we add the content of the 10mL from the colony picking.

Results

Before the IPTG induction we obtained OD₆₀₀ that were between 0.6 and 0.8, which is the required range at PTSP to pursue with the induction. Then, we measured the OD₆₀₀ after the IPTG induction, and observed small values to the ones PTSP usually gets after induction (between 2 and 10), but it's sufficient to do the purification.

Analysis In the gel we ran to see if there is the expression of our protein, we could see a small band around 100kDa, which is higher than the expected size of the silk (around 70 kDa). However, since we obtained good OD₆₀₀ values, we decided to do the purification. Indeed, it can be that the silk protein is present but in very small amounts and concentration. To check this assumption we should do the purification and run an SDS-page gel as well as a Western Blot, and see if anti-His antibodies recognize the protein of interest.

We will do a protein purification using the purification protocol of PTSP.

Aim The aim of this experiment is to purify our new 01a (mSA-silk-CBD) construct. To do so, we will use the Ni-NTA affinity beads and columns of the Protein Facility (PTSP). This purification method is used to purify His-tagged proteins. Unbound proteins will be washed away with a specific washing buffer, and we will purify our protein of interest with specific elution buffers that contain increasing concentration of imidazole. We make all the buffers ourselves.

Protocol Protein purification PTPSP

Remarks

• We diluted the pellet into 30mL of wash buffer A.

• We added 500 μl of lysozyme to better lysate the cells.

• We used a plastic disposable column to wash the beads since the centrifuge was full.

• We mixed the supernatant with the beads for 2 hours and not overnight.

• We did simply 2 elution steps: elution 1 at 250mM imidazole and elution 2 at 500mM imidazole.

Results

Analysis Even if it looks like we have proteins in elution 1, we need to do a quality control (SDS page or WesternBlot) to check whether we actually purified our protein of interest or if the measured concentration refers to a contaminant protein.

Aim The aim of this experiment is to run the SDS-PAGE of our samples from the silk protein purification, to see if we can find our protein of interest.. By the end of this experiment, we should choose the tube with the higher amount of protein, measure the purity and concentration of the protein and use it for the coating of the aerogel.

Protocol SDS-PAGE

Remarks

• We put 30 μL of samples and 10 μL of Tris-Glycine SDS sample buffer (2X).

• We loaded 3 μL of ladder, 5 μL of supernatant and flow through and 10 μL of all the other samples.

• We run the gel (stained with Coomassie Blue Protein Stain) for 30 min at 180 V.

Results

Analysis Since we obtained the wanted plasmid, we transformed new BL21(DE3) competent cells to start a new protein expression and purification. By doing a gel comparing the protein expression before and after IPTG induction (fig 18.A), we could see a band around 100 kDa. Indeed, in the SDS-PAGE gel (fig 18.B), there is a band around 100 kDa in both the washes and both the elutions, in addition to some contaminant proteins. Even if we expected the protein to be around 74kDa, this 100kDa lane should correspond to our new silk fusion protein, since we obtained similar ones in the other purifications. By measuring the concentration (0.37 mg/mL), we obtained a final amount of 11mg of the silk fusion protein (01a).

Aim To transform E. Coli BL21(DE3) competent cells with our construct 01a (mSA-silk-CBD), express the corresponding protein and purify it. Overall, here we want to retrieve our induced Silk (4 repetitive modules) fused to mSA, CBD and His tag protein. We will use the plasmid 01a sent by Genescript with the added sites in the N terminus of the fusion protein, since we were not able to produce the proteins from the cloned plasmid without those sites. To do so, we went to the protein facility to redo the purification protocol from 24.08.2022.

Aim To do the transformation of E.Coli Competent Cells using BL21(DE3) strain, with the 01a plasmid (mSA+Silk(4X)+CBD). We will plate the cells on a LB-agar plate containing Kanamycin, and we expect the cells that have integrated our plasmid to resist those antibiotics.

Protocol Bacterial transformation

Remarks

• We didn’t have gas anymore so we couldn’t work with the flame, therefore we worked under the laminar flow hood for sterile techniques.

• For positive control, the plasmid “Tomato” from the Summer School was used. We took 3 μl of a sample with 15 ng/μl of concentration.

• Cells were incubated on ice for about 10 min before heat shock.

• 400 μl of SOC medium was added after the heat shock.

• 100 μl of transformation reaction was plated on LB-Kana plates and the 300 μl left were put in 50 ml of LB-Kana in an Erlenmeyer to do liquid transformation cultures overnight at 37°C under shaking (200rpm).

Results

After the overnight incubation, we observed the presence of colonies in the BL21(DE3) transformed cells plates of 01a and the positive control, no colonies were observed on the negative control, as expected.

After the overnight incubation of the 01a liquid transformation culture, an OD₆₀₀ of 2.914 was reached the following morning.

Analysis The 01a GeneScript plasmid transformation worked well for BL21(DE3) since we can observe colonies on the LB-Kanamycin plates and the OD₆₀₀ of the transformation liquid culture is high, meaning that many bacteria were able to grow.

Since the transformation was successful, we can use the transformation liquid culture for culture upscaling.

Aim The aim of this experiment was to grow the BL21(DE3) competent cells transformed with the new 01a plasmid in a TB medium with Kanamycin to prepare big cultures and then to induce the transcription with IPTG of the plasmid 01a (mSA-N[AS]4C-CBD-10xHis).

Protocol IPTG induction

Results OD₆₀₀ measurements before and after IPTG induction are below :

Before the IPTG induction we obtained OD₆₀₀ that were between 0.6 and 1, which is the required range at PTSP to pursue with the induction. Then, we measured the OD₆₀₀ after the IPTG induction, and observed small values to the ones PTSP usually gets after induction (between 2 and 10), but it's sufficient to do the purification. We noted that the 01a culture 1 had an unusually low OD₆₀₀ of 0.45.

Analysis Considering the low OD₆₀₀ of 0.45 of 01a culture 1, we decided to purify the cultures 1 and 2 together by resuspending the pellets in the same tube.

Aim The aim of this experiment is to purify our induced mSA-silk-CBD-10xHis protein. To do so, we will use the Ni-NTA affinity beads and columns of the Protein Facility (PTPSP). This purification method is used to purify His-tagged proteins. Unbound proteins will be washed away with a specific washing buffer, and we will purify our protein of interest with specific elution buffers that contain increasing concentration of imidazole. We make all the buffers ourselves.

Protocol Protein purification PTPSP

Remarks

1. Since the first 01a culture had a much lower OD than the second 01a culture, we decided to merge the two pellets in the purification process.

2. We diluted the total pellet into 35mL of wash buffer A.

3. We added 175 μl of lysozyme to better lyse the cells.

4. We mixed the supernatant with the beads for 2 hours and not overnight.

5. Wash 2 was done with 70mM (50mL per column) instead of 50mM (30mL) to remove more unspecific binding before the elution.

6. We did simply 1 elution step at 50 mM EDTA (pH 8) in 20mL, which elutes the nickel ions of the Ni-NTA beads with our His-tagged proteins, instead of 2 elutions in 250mM and 500mM imidazole, because of the issues we faced for the previous purification with our proteins containing a 6xHis-tag on the N terminus and a 10xHis-tag on the C terminus.

7. After the purification, we put the elution of 01a in the dialysis buffer (same as wash buffer A) overnight. This step is supposed to remove the EDTA and the nickel ions.

8. We did not concentrate our proteins because the concentration measured after dialysis was enough for our next experiments.

Results We calculated the amount of the protein after purification by measuring the absorbance at 280 nm with the Nanodrop:

The values were obtained based on the absorbance at 280 nm, the molar extinction coefficient and the molecular weight of the protein 01a that were plugged in the protein model used to determine the amount of proteins needed to coat our aerogels.

Aim The aim of this experiment is to run the SDS-PAGE of our samples from the silk protein purification, to see if we can find our protein of interest and in which tube. By the end of this experiment, we should choose the tube with the higher amount of protein (hopefully in the elution), measure the concentration of the protein and use it for the coating of the aerogel.

Protocol SDS-PAGE

Remarks

1. We put 30 μL of samples and 10 μL of Tris-Glycine SDS sample buffer (2X).

2. We load 5 μL of ladder, 10 μL of supernatant and flow through and 15 μL of all the other samples.

3. We run the gel for 60 min at 100 V.

Results

In the elution lane, two distinct bands can be observed, one around 100-120 kDa and one around 75 kDa. Both bands can also be observed in the wash 2 lane, albeit with a lower intensity.

Analysis The band observed in the elution lane around 100-120 kDa is consistent with our previous purification of 01a. We can therefore assume that it is our expected silk protein. The band observed around 75 kDa was also present in the 01b and 03a SDS-PAGEs, all three purifications and quality controls were done the same day, so there is a possibility that it is a new contaminant protein.

If the protein observed around 100-120 kDa is our expected silk, then it should also appear in the western blot. As for the protein around 75 kDa, we can also assess if it is a contaminant with a western blot.

Aim To do a Western Blot of the gel of the 01a construct (mSA-silk-CBD-10xHis). We will use His-tag antibodies that bind the same His tag we used for purification in order to identify specifically the purified silk protein and see in which sample it is the most present.

Protocol Western Blot

Results

In the wash 1 and elution lanes, bands can be observed around 100-120 kDa, similar to the SDS-PAGE.

Analysis Looking at the wash 1 and elution lanes, we can observe similar bands around 100-120 kDa. These are analogous to the previously obtained SDS-PAGE. The sizes are coherent with the ones obtained in our previous 01a purifications. In the first three lanes (lysate, supernatant, flow through) the bands observed around 15 kDa can be attributed to the lysosome. The very large bands on top can be attributed to the usually high amount of proteins loaded in the gel.

The proteins observed in the wash 1 and elution lanes correspond by size and by anti-His antibody recognition to our protein of interest. We have therefore purified our 01a (mSA-silk-CBD) construct and will be able to use it in future experiments. We will proceed with tests on our hydrogels, aerogels, as well as silk biofilm creation tryouts.

Aim The aim of this experiment is to produce a silk biofilm inspired by the work of Felix Bauer1, in order to increase the hydrophobicity of our recombinant silk protein (mSA-Silk-CBD). The final goal would be to coat our cellulose aerogel with a silk biofilm to make it waterproof.

Protocol Silk biofilm

Remarks

• 01a We have a concentration of 0.715 mg/ml for 01a silk protein. We want to use 2.5 mL of 01a protein solution, which corresponds to m_silk = 0.715 mg/ml x 2.5 mL = 1.7875 mg. We want a 1% (w/v) solution of silk protein in formic acid, we need the following volume of formic acid: V_formic_acid = 100 [μl/mg] x m_silk [mg] = 178.75 μl → We rounded up to 179 μl.

• Strong safety guidelines needed to be followed when doing the experiment as we use formic acid in our production process. All manipulations were performed under a chemical fume hood and we wore a lab coat, goggles, special gloves really thick and a facial mask.

Results

Analysis On (A), one can observe some crystals and liquid. On (B), one can still observe some crystals but no more liquid seems to be present. The crystals might be formed by the mixture of our proteins in liquid form with the formic acid. It does not have a big impact on the final properties of the biofilm. Our hypothesis regarding the presence of liquid in the Petri dishes is that the biofilm did not dry enough. The fact that the modeling of the silk proteins showing a 3D structure with beta-sheets and beta-helices confirmed us that our proteins should form a hydrophobic biofilm. Indeed, their particular folding suggest hydrophobicity, which is a good sign for the hydrophobicity of the biofilm. We decided to let it dry more for one day. At the end, there was no more liquid but we did not manage to peel off the biofilm from the Petri dish. Our hypothesis is that our biofilm was too dried after two days of drying.

Conclusion We managed to form a biofilm with our recombinant silk protein (mSA-silk-CBD). The next step would be to test its hydrophobicity. For our next trial of doing a biofilm, we will try to let the biofilm dry in less than 2 days to see if we manage this time to peel it off the Petri dish.

Aim To produce a silk biofilm inspired by the work of Felix Bauer2, in order to increase the hydrophobicity of our recombinant silk protein (mSA-Silk-CBD). The final goal would be to coat our cellulose aerogel with a silk biofilm to make it waterproof. We are doing this experiment for the second time, in order to improve the evaporation time and reduce the biofilm’s stickiness on the petri dish. We will therefore try 3 different protocols for each silk biofilm : the first one with an evaporation’s time of 24 hours, the second one with an evaporation’s time of 48 hours, both with polystyrene petri dishes and the third one with a glass petri dish and an evaporation’s time of 24 hours. Moreover, we will do the same protocol for the GFP fusion protein to have a proper control for this experiment.

Protocol Silk biofilm

Remarks

• 01a We have a concentration of 0.0928 mg/ml for 01a silk protein. We want to use 2.5 mL of 01a protein solution, which corresponds to m_silk = 0.0928 mg/mL x 2.5 mL = 0.232 mg. We want a 1% (w/v) solution of silk protein in formic acid, we thus need the following volume of formic acid: V_formic_acid = 100 [μl/mg] x m_silk [mg] = 23.2 μl → We rounded up to 24 μl.

• 03a We have a concentration of 0.0942 mg/ml for 03a GFP protein. We want to use 2.5 ml of 03a protein solution, which corresponds to m_gfp = 0.0942 mg/ml x 2.5 ml = 0.2355 mg. So if we want a 1% (w/v) solution of gfp protein in formic acid, we need the following volume of formic acid: V_formic_acid = 100 [μl/mg] x m_gfp[mg] = 23.55 μl → We rounded up to 24 μl.

• Strong safety guidelines needed to be followed when doing the experiment as we use formic acid in our production process. All manipulations were performed under a chemical fume hood and we wore a lab coat, goggles, special gloves really thick and a facial mask.

• We did not take pictures of the result for the glass Petri dishes because they were similar to the polystyrene Petri dishes.

Results

Analysis On figure 23.A and 23.B, we show the silk fusion protein biofilm. We can clearly see some cracks which indicate that we dried the biofilm too much. Even if it shouldn’t be a problem to test the hydrophobicity, it could become one to remove the biofilm from the dish. Indeed, as for the first experiment it was impossible for us to take the biofilm out, even by cutting the edges of the petri dish. On figure 1.C and 1.D, we show the GFP fusion protein biofilm that we did as a control for the experiment. Here, we can clearly see that the GFP made some crystals and cubic structures, so that the intended biofilm is less homogeneous than the silk one. This result indicates that it is not possible to make a biofilm from any proteins, and in this case, we didn’t obtain a proper one.

Conclusion We managed to form a biofilm with our recombinant silk fusion protein (mSA-silk-CBD), but not with the GFP fusion protein (mSA-GFP-CBD). With these biofilms, we will be able to do an hydrophobicity test and compare the results.

Aim The aim of this experiment is to show that the silk fusion protein biofilm is hydrophobic. To do so, we prepared a silk fusion protein biofilm (01a) as well as a GFP fusion protein biofilm (03a), and we added droplets of water on top of it. We first measured the angle of the water droplet by putting the microscope at 70°, to assess the intensity of the capillary forces on the material 3 and the surface wettability. Then we measured the time it takes for the water to be absorbed, computed the mean values for each biofilm and compared them to conclude about hydrophobicity.

Protocol Keyence microscope VHX-7000

Remarks

• The first measurements were conducted on the silk fusion protein (01a) and GFP fusion protein (03a) biofilms with only one droplet of water to measure the contact angle of the water with the biofilm.

The equation of interest was given by :





• As shown in the above figure, we knew that different materials have different angles in contact with water and we could measure the hydrophobicity of our material with this specific measure. The higher the angle the better is the hydrophobicity of the material.

• The second measurements were conducted on the silk and GFP biofilms with three water droplets for each sample to assess the drying time of the water.

Results

Analysis Following the above equation and (fig. C and D), we could see that the silk fusion protein biofilm (01a) had a higher contact angle than the GFP biofilm. Moreover, by measuring the time it takes for the water to be absorbed (fig. E and F), we obtained a mean of 466,33 s for the silk biofilm, and 28,33 s for the GFP biofilm. With these values, we can see that it takes more time for a drop of water to be evaporated or absorbed from the silk biofilm. The following results suggest that our biofilm would help protect the aerogel from water droplets. The measurements would need to be redone in a statistical manner and we should definitely try to concentrate more on the silk content of the fusion protein to see an improvement of the droplet. Moreover, we noticed clearly that between the two biofilms in (fig. E and F) that the droplets between the two biofilms had different shapes. They were wieder for the GFP sample compared to the Silk one. The following confirmed clearly the enhancing of hydrophobicity by our Silk biofilm.

Aim To express our 01b (SR-Avitag) construct in BL21(DE3) and then purify the obtained protein.

Aim To transform BL21(DE3) cells with our 01.b plasmid (SR-Avitag plasmid). We used as positive control a plasmid containing a resistant gene to ampicillin (pUC19 control), and as negative control water to model the absence of plasmid. We expect to observe grown colonies from the positive control, and the absence of colonies with the negative control, since the bacteria will be treated with antibiotics.

Protocol Bacterial transformation

Results After the overnight incubation, the negative control presented no colony, the positive presented some colony, and finally the 01b transformed plate presented numerous colonies.

Analysis The negative control worked as expected (A). No plasmid were uptaken by bacteria, thus no resistance was obtained. The positive control worked as expected (B). Less resisting colonies were observed because the Ampicillin plasmids’ concentration was lower than the one of the Kanamycin plasmids. Finally, the 01b plasmid transformation went well (C). Many colonies were observed, with low satellite colonies, which is good.

Aim To amplify the plasmid 01b by picking a bacterial colony after bacterial transformation.

Protocol Colony picking

Results We picked the plates from the 01b culture of bacteria from the transformation plate we made on 18.07.2022. We can measure OD₆₀₀ measurements of the culture on the next day to be sure that bacteria have grown overnight.

Aim To grow the bacteria transformed with the plasmid 01b in a LB medium with Kanamycin to prepare big cultures. The next day we induced the transcription of the plasmid 01b containing SR fused to Avitag and His tag for protein expression with IPTG.

Protocol IPTG induction

Remarks

• For the bacterial inoculation, we did three falcon tubes to induce several IPTG concentrations the next day. We forgot to turn on the flame when pipetting the LB medium so we needed to redo the three falcon tubes. (LB medium bottle has been labeled to wait one week to see if there is any bacterial development within the bottle). We added 10 uL of Aral’s bacterial culture without shaking the tube softly and we noticed that the bacteria were at the bottom of the tube. We decided to add 10 uL of the bacterial culture in addition after shaking the falcon tube softly. ⇒ to avoid this, we can let the bacterial culture at room temperature.

• For the IPTG induction, we did 4 IPTG induction samples with 3 different concentrations (0.5 mM (1 μl) → one sample (1), 1 mM (2 μl) → two samples (3 and 3’), 2 mM (4 μl) → one sample (2)). We did 2 control samples with no IPTG induction from the inoculated cultures 2 and 3.

Results Our first OD₆₀₀ measurements results were too high, so we decided to do a 4:5 dilution to obtain values in the good range (0.4-0.6). For each dilution we took 2 ml of the bacterial culture and 0.5 ml of medium (9ml of LB and 90μl of kanamycin) to obtain a 4:5 dilution.

After 3 hours of incubation with IPTG, we measured the OD₆₀₀ again. The cell culture should have a final OD₆₀₀ < 6.0 for efficient processing and purification. We don’t have OD₆₀₀ measurements for the two controls since we did not have 1 ml to use only for the measurements.

Aim To purify plasmid DNA from our overnight culture of bacteria transformed with plasmid 01b, then send it to the sequencing center, to confirm that plasmid 01b was taken up by bacteria and that there is no mutation within the DNA sequence.

Protocol MiniPrep

Results

All sequencing signals of each sample showed the same pattern with high noise and no sequence could be determined.

Analysis The concentrations obtained for the NanoDrop are quite low for most of the samples, it is barely enough for the sequencing (concentration range required: 40-100 ng/μl). The purity of the samples (A260/A280 ratio) varies among samples, but surprisingly samples with low concentrations seem to have a better purity than the high concentrated ones.

Sequencing results (Figure 1) show an important contamination of our samples as we can tell by the high noise background. Indeed, we can observe peaks of different colors at the same position, where there should be only one.

This can be due to several sources:

• Water from our lab could be contaminated

• Pipettes from our lab could be contaminated

• The original plasmid 01b sent by the company could be contaminated

• The primers from Danica’s lab could be wrong or contaminated

Discussion Out of the 12 Minipreps, 6 were sent for sequencing, with only T7Fw primer, not the T7Rv. The concentrations are low but we can still work with. These purified plasmid DNA samples don’t seem to be completely pure. We carry out a second round of sequencing to determine the source of error, so that we can continue to move forward.

Aim To determine the source of our contaminated sequencing results.

We sent the following samples for sequencing, using this time the T7 primer from Microsynth’s standard primer list:

• Original plasmid 01b from GeneScript

• Water from our laboratory

• Water from another laboratory, known to be pure

• Three of the samples already sent for sequencing (PV, CN, Extra)

We expect the following results :

Protocol MiniPrep

Results Sequencing alignment file

Analysis Original 01b plasmid and sequenced 01b plasmids from the other samples. (A) Original 01b plasmid designed in SnapGene. (B), (C), (D), (E) are the sequenced plasmids from the other samples. It shows an intact open reading frame, as well as every necessary component. However, they all show the same NcoI restriction site, 6x His and thrombin site insertion. They also show no PmeI restriction site. In total 102 bp were added.

Since all the plasmids show the same insertion, we can conclude that the NcoI restriction site, a 6xHis-tag, a thrombin site and a T7 tag were part of the company’s pet28A’s backbone and don’t correspond to our original design.

Discussion These additional sites, and especially the second His tag might interfere with the purification of our proteins. We can continue our expression and purification with these plasmids and in parallel try to get rid of the inserted site for optimization.

Aim To run the SDS page of our samples from the SR+avitag protein purification, to find in which tube we can find most of our eluted protein of interest. By the end of this experiment, we should choose the tube with the higher amount of protein, measure the purity and concentration of the protein and use it for the coating of the aerogel.

Protocol SDS-PAGE

Results After the protein staining buffer step, we were not able to see our protein of interest on the gel, so we washed it with water twice while keeping it on a shaker. Then we left the gel overnight in water on a shaker.

Analysis While the ladder, the lysate and the washes are visible, no bands were observed in the elution lanes. This suggests that either the elutions contained no protein, or that the many mishaps while doing the gel caused the samples to spill. It could also be the very low amount of sample that was put in each lane (only 2.5 μL). The stain used for the gel (Imperial Protein Stain) proved to be very strong as it left a lasting purple color on the gel which created a gray background when using the Illuminator.

Conclusion No protein could be observed with this SDS-PAGE gel. For clearer pictures and protein visualization, a different protein stain should be used. The next step is to redo an SDS-PAGE to troubleshoot why

Aim To run the SDS page of our samples from the SR-Avitag protein purification, to find in which tube we can find most of our eluted protein of interest. By the end of this experiment, we should choose the tube with the higher amount of protein, measure the purity and concentration of the protein and use it for the coating of the aerogel.

Protocol SDS-PAGE

Results

Analysis Our protein of interest seems to appear in every sample around 50-55 kDa, which is consistent with Mingdao iGEM 2015’s observations of the SR protein at 53kDa. It can be considered that the added parts did not greatly influence the overall weight, therefore our protein’s weight should be similar to 53kDa. Stronger lanes in elution are observed for sample 2 (figure 1.B), which suggests that a 2 mM concentration of IPTG for induction gives a better yield of our protein. Using 0.5M or 0.75M Imidazole for the second elution does not seem to affect the yield of our protein. Some contamination can be observed in most of the samples, especially in the Elution 1 lanes and around 15kDa. Further analysis of our 01b plasmid might allow us to determine which protein it could be. The Imperial Protein Stain used also left a lasting purple color on the gels which made the imaging difficult by creating a gray background.

Conclusion A protein of corresponding weight was observed in each sample. A consistent contaminant was also seen in the elution lanes around 15kDa. Redoing the SDS-PAGE gels while using a different stain and purer samples might give better results. The next step is to do a Western Blot on the gels where we can observe proteins around 53kDa in the elution lanes. It would confirm that it is indeed the one we are trying to express.

Aim To run a Western blot with a His-tag antibody to determine if the protein observed in the elution is indeed the protein we wanted to express.

Protocol Western Blot

Results

Analysis It could be that we washed away our protein of interest during the step where we had to take off the Ponceau with PBS + Tween. Indeed, after the addition of Ponceau we were able to see lines in the lysate, the Wash 1 and the elution. But by washing Ponceau the protein that appeared in the elution line wasn’t visible anymore. The absence of signal in the elution line can also be explained by mistakes during the protein purification, since we see a signal in the lysate and the wash 1.

Conclusion We could confirm the presence of our SR+Avitag protein during the Ponceau step, since it corresponds to the good size. However, it seems that the protein is only visible in the lysate and wash 1 and not in the elutions. We need to trouble-shoot our purification method because if our protein of interest is only present in the lysate and the wash1, it means that our purification did not work well.

Aim The aim of this experiment sequence is to transform competent cells with our construct, express the corresponding protein and purify it. Overall, here we want to retrieve our induced SR protein fused to Avitag and His-tag, to then make it bind the silk biofilm that will coat the aerogel. To do so, we went to the protein facility to test a new purification protocol and new bacterial strains.

Aim The aim of this experiment is to do the transformation of E.Coli Competent Cells using two different strains, BL21(DE3) and Rosetta, with the 01b plasmid (SR + Avitag). We will plate the cells on LB-agar plates containing Kanamycin, and we expect the cells that have integrated our plasmid to resist this antibiotic.

Protocol Bacterial transformation

Results After the overnight incubation, we observed the presence of colonies in both the Rosetta and the BL21(DE3) transformed cells plates but there were more colonies on the BL21(DE3) plate. The negative control plate did not show any colony, and the positive control plate was full of colonies, as expected.

After the overnight incubation, we observed the presence of colonies only in the BL21(DE3) transformed cells plate, but not in the Rosetta transformed cells plate. The negative control plate did not show any colony, and the positive control plate was full of colonies, as expected.

Analysis The 01b plasmid transformation worked well for BL21 since we can observe some colonies on the Kanamycin plates, but not for Rosetta. The bacterial transformation of 01b worked for BL21 strain, so we can pursue the process by doing colony picking in a sterile environment (with a flame).

Aim The aim of this experiment was to grow the BL21 bacterial strains transformed with the plasmid 01b in a TB medium with Kanamycin to prepare big cultures and then to induce the transcription with IPTG of the plasmid 01b containing the fire resistance protein SR fused to Avitag and His tag.

Protocol Colony picking

Remarks We used 2 samples for this experiment since colonies grew from the two bacterial strains: rosetta and BL21.

Results

Before the IPTG induction we obtained OD₆₀₀ that were between 0.6 and 0.8, which is the required range at PTSP to pursue with the induction. Then, we measured the OD₆₀₀ after the IPTG induction, and observed small values to the ones PTSP usually gets after induction (between 2 and 10).

Analysis In the gel we ran to see if there is the expression of our protein, we could see the expression of the fire resistance protein from the 01b plasmid. Indeed, even if we obtained smaller OD₆₀₀ values than the ones PTSP is used to obtain after the IPTG induction (OD₆₀₀ between 2 and 10), we decided to do the purification.

Aim The aim of this experiment is to purify our induced fire resistance protein fused to Avitag and His tag, from initiale samples coming from BL21. To do so, we will use the Ni-NTA affinity beads and columns of the Protein Facility (PTSP). This purification method is used to purify His-tagged proteins. Unbound proteins will be washed away with a specific washing buffer, and we will purify our protein of interest with specific elution buffers that contain increasing concentration of imidazole. We make all the buffers ourselves.

Protocol Protein purification PTPSP

Remark

• Still our control (GFP fused with CBD and mSA) looks to be stuck on the beads even after the second elution, we increased the imidazole concentration and did other elutions until reaching the stock concentration of 5M.

• We need to be careful now, because imidazole can be aggressive for the stability of proteins, and may enhance their denaturation. We thus need to find solutions to avoid this required increasing amount of imidazole in the elutions samples.

Results We calculated the amount of the protein by measuring the concentration with the nanodrop. First we measured it right after the purification, when we concentrated the proteins using a filter, so just before to put in dialysis. Then we measured it after the dialysis and finally after the centrifugation that followed the dialysis.

Analysis After the purification, we concentrated the proteins using a 30kDa filter, then quantified the amount of proteins we obtained and obtained a relatively low amount. Then we put the proteins under dialysis, and quantified again the amount after the dialysis. At this point, strangely it looked like we had more protein, which does not make sense. To verify that this increase in quantity is not a bias of the nanodrop due to the absorbance of protein aggregations, we decided to centrifuge our proteins. In this way if a pellet appears, it means that there is indeed an aggregation which may mean that the proteins have been denatured. Unfortunately, we observed a pellet which means that our protein is certainly no more functional. Moreover, we quantified again the amount and we realized that we lost most of the proteins. This is certainly due to the solvent exchange during the dialysis that was too intense and may have denatured the proteins. For the next purification, we will have to find other dialysis buffers to improve the yield of protein we obtain at the end of the purification.

Aim The aim of this experiment is to run the SDS-PAGE of our samples from the SR protein purification of samples coming from BL21, to find in which tube we can find most of our protein of interest. By the end of this experiment, we should choose the tube with the higher amount of protein, measure the purity and concentration of the protein and use it for the binding with the silk biofilm.

Protocol SDS-PAGE

Remarks

• We have two different supernatant samples because we did the sonication step of the purification twice since the control had a very green pellet even after the first lysis. This meant that not all cells were lysed during the first sonication, so we did it a second time to recover more proteins.

• We have no elution 3 (E3) here because we first increased the concentration to elute the control, and saw that the elution 3 wasn’t working so well to remove the green color of the beads. Thus we decided to not do this step in the other samples.

Results

Analysis In the gel, we can see a band around 38kDa in elution 4, which is the expected size of our SR fire resistance protein. Strangely we do not see any band in the firsts elutions, which means that this protein has a really strong affinity with the beads. However, since we see a line only in elution 4, it means that we found the right imidazole concentration to optimally elute the protein. During the protein purification, we had to elute the protein with a high amount of imidazole which can damage it. Therefore, we dialysed our SR protein to get rid of this imidazole excess. Unfortunately, after this dialysis step we lose most of the proteins so we have to find another way to remove the imidazole for the next purification.

Aim The aim of this experiment is to do a Western Blot with the elution samples to see whether the protein we see around 38kDa is our SR protein that we hope we successfully purified. We used the samples from the pellet of 01b BL21, the supernatant 2 of 01b BL21, and the elutions: E1, E2, E4 and E5.

Protocol Western Blot

Results

We wanted to concentrate them, so we used a filter of 30kDa. And then to remove the excess of imidazole by doing a dialysis. Unfortunately, after this dialysis step we lose most of the proteins so we have to find another way to remove the imidazole for the next purification.

Analysis Here we clearly see a band around 38kDa in elution4. This means that a protein has been recognized by the anti-His antibodies. It allows us to confirm that this protein is indeed our SR protein. However, we didn’t obtain a really good amount of it and we lost most of the protein in the dialysis step. Even if we lose most of the proteins, the overall result remains promising since we successfully purified this fire resistance protein. For the next purification, we will have to figure out how to concentrate the protein and remove the excess of imidazole without losing the proteins.

Aim The aim of the following experiments is to transform competent cells with our new construct, express the corresponding protein and purify it. Overall, here we want to retrieve our induced SR protein (fire resistant) fused to avitag and His tag that do not contain the added site of Gene Script. To do so, we went to the protein facility to redo the purification protocol from 24.08.2022.

AimThe aim of this experiment is to do the transformation of BL21 E.Coli Competent Cells for protein expression with the new 01b plasmid (SR protein + avitag) obtained after the miniprep of the 8th of september. We will plate the cells on agar plate containing Kanamycin, and we expect the cells that have integrated our plasmid to resist those antibiotics.

Protocol Bacterial transformation

Remarks

• Here we used 25 μl of E.Coli competent cells and 2.5 μl of the new 01b plasmid (SR + Avitag).

• We didn’t have gas anymore so we couldn’t work with the flame, therefore we worked under the laminar flow hood for sterile techniques.

• We didn’t do any controls because we didn’t have enough plates and we already did many transformation controls.

• We used 200 μl of SOC medium instead of 100 since we started with more cells.

Results

Analysis The 01b plasmid transformation of BL21 competent cells worked well : we can observe colonies on the Kanamycin plates. The bacterial transformation of the new 01b worked, so we can pursue the process by doing colony picking in a sterile environment.

Aim The aim of this experiment is to amplify the plasmids 01b by picking bacterial colonies from bacterial transformation made on 09/09 and to grow bacteria in bigger liquid cultures.

Protocol Colony picking

Remarks

• We put 10 mL of LB-Kana medium in a 15 mL falcon tube.

• We incubated the cultures for about 20h.

Aim The aim of this experiment was to grow the BL21 competent cells transformed with the new 01b plasmid in a TB medium with Kanamycin to prepare big cultures and then to induce the transcription with IPTG of the plasmid 01b containing SR protein fused to avitag and His tag.

Protocol IPTG induction

Remarks We directly did 1.3L of big cultures. So we put 1.3L of TB+Kana and we add the content of the 10mL from the colony picking.

Results

Before the IPTG induction we obtained OD₆₀₀ that were between 0.6 and 0.8, which is the required range at PTSP to pursue with the induction. Then, we measured the OD₆₀₀ after the IPTG induction, and observed small values to the ones PTSP usually gets after induction (between 2 and 10), but it's sufficient to do the purification.

Analysis In the gel we ran to see if there is the expression of our protein, we couldn’t see any relevant expression. However, since we obtained good OD₆₀₀ values, we decided to do the purification. Indeed, it can be that the SR protein is present but in very small amounts and concentration. To check this assumption we should do the purification and run an SDS-page gel as well as a Western Blot, and see if anti-His antibodies recognize the protein of interest.

We will do a protein purification using the purification protocol of PTSP.

AimThe aim of this experiment is to purify our induced SR protein fused to avitag and His tag protein. To do so, we will use the Ni-NTA affinity beads and columns of the Protein Facility (PTSP). This purification method is used to purify His-tagged proteins. Unbound proteins will be washed away with a specific washing buffer, and we will purify our protein of interest with specific elution buffers that contain increasing concentration of imidazole. We make all the buffers ourselves.

Protocol Protein purification PTPSP

Remarks

• We diluted the pellet into 30mL of wash buffer A.

• We added 500 μl of lysozyme to better lysate the cells.

• We used a plastic disposable column to wash the beads since the centrifuge was full(separation by gravity).

• We mixed the supernatant with the beads for 2 hours and not overnight.

• We did simply 2 elution steps: elution 1 at 250mM imidazole and elution 2 at 500mM imidazole.

Results

Analysis Even if it looks like we have proteins in elution 1 and 2, we need to do a quality control (SDS page or WesternBlot) to check whether we actually purified our protein of interest or if the measured concentration refers to a contaminant protein.

AimThe aim of this experiment is to run the SDS-PAGE of our samples from the SR protein purification, to see if we can find our protein of interest and in which tube. By the end of this experiment, we should choose the tube with the higher amount of protein, measure the purity and concentration of the protein and use it for the coating of the aerogel.

Protocol SDS-PAGE

Remarks

• We put 30 μL of samples and 10 μL of Tris-Glycine SDS sample buffer (2X).

• We load 3 μL of ladder, 5 μL of supernatant and flow through and 10 μL of all the other samples.

• We run the gel (stained with Coomassie Blue Protein Stain) for 30 min at 180 V.

Results

Analysis For the SR fusion protein, even if we couldn’t see a significant protein expression after the IPTG induction (fig 11.A), we did the purification of the protein. In the SDS-PAGE gel (fig 11.B), we can see a band around 70kDa in the elution 1, in addition to several contaminant proteins at different sizes. As we expected a band around 36.4kDa, this band (and all the other ones) is a contaminant protein. This means that we didn’t successfully purify our new SR fusion protein.

Here we cannot really conclude if we obtained our protein of interest, but it is certainly not the case. To confirm its presence, we could do a western blot. If the anti-his binds the corresponding band, then it is most likely that we purified our protein. However, since we did the purification of 01a and 03a at the same time, and that it was a failure, we decided to redo the purification of the 3 constructs using the old plasmid since it worked once.

Aim To transform competent cells with our construct, to express the corresponding protein and purify it. Overall, we want to retrieve our induced SR protein fused to avitag and His tag. We will use the plasmid sent by Genescript with the added site, since we were not able to produce the proteins without it. To do so, we went to the protein facility to redo the purification protocol from 24.08.2022.

Aim To do the transformation of E.Coli Competent Cells using the BL21(DE3) strain, with the 01b plasmid (SR+Avitag). We will plate the cells on a LB-agar plate containing Kanamycin, and we expect the cells that have integrated our plasmid to resist those antibiotics.

Protocol Bacterial transformation

Remarks

• We didn’t have gas anymore so we couldn’t work with the flame, therefore we worked under the laminar flow hood for sterile techniques.

• For positive control, the plasmid “Tomato” from the Summer School was used. We took 3 μl of a sample with 15 ng/μl of concentration.

• Cells were incubated on ice for about 10 min before heat shock.

• 400 μl of SOC medium was added after the heat shock.

• 100 μl of transformation reaction was plated on LB-Kana plates and the 300 μl left were put in 50 ml of LB-Kana in an Erlenmeyer to do liquid transformation cultures overnight at 37°C under shaking (200rpm).

Results After the overnight incubation of the 01b liquid transformation culture, an OD₆₀₀ of 2.766 was reached the following morning.

After the overnight incubation, we observed the presence of colonies in the BL21(DE3) transformed cells plates of 01b and the positive control, no colonies were observed on the negative control, as expected.

Analysis The 01b (SR+Avitag) GeneScript plasmid transformation worked well for BL21(DE3) since we can observe colonies on the LB-Kanamycin plates and the OD₆₀₀ of the transformation liquid culture is high, meaning that many bacteria were able to grow. Since the transformation was successful, we can use the transformation liquid culture for culture upscaling.

Aim To grow the BL21(DE3) competent cells transformed with the 01b (SR+Avitag) plasmid in a TB medium with Kanamycin to prepare big cultures and then to induce the transcription with IPTG of the plasmid 01b (SR-Avitag-10xHis protein).

Protocol IPTG induction

Remarks

• Since the transformation was successful, we directly used the 50mL of transformation liquid culture (OD of 2.766) to make bigger cultures.

• We did 1L of big cultures with 25mL of transformation liquid culture in 1L of TB+Kana.

Results

Analysis Before the IPTG induction we obtained OD₆₀₀ that were between 0.6 and 1, which is the required range at PTSP to pursue with the induction. Then, we measured the OD₆₀₀ after the IPTG induction, and observed a normal value for the culture 2 of 01b, but an extremely low value for the culture 1 of 01b (the one cultured with biotin) indicating that this culture died, so we could only do a purification with the culture 2 without biotin.

Aim To purify our induced SR-Avitag-10xHis protein. To do so, we will use the Ni-NTA affinity beads and columns of the Protein Facility (PTSP). This purification method is used to purify His-tagged proteins. Unbound proteins will be washed away with a specific washing buffer, and we will purify our protein of interest with specific elution buffers. We make all the buffers ourselves.

Protocol Protein purification PTPSP

Remarks

• Since the 01b culture with biotin died, we only purified the 01b culture without biotin.

• We diluted the total pellet into 35mL of wash buffer A.

• We added 175 μl of lysozyme to better lyse the cells.

• We mixed the supernatant with the beads for 2 hours and not overnight.

• Wash 2 was done with 70mM (50mL per column) instead of 50mM (30mL) to remove more unspecific binding before the elution.

• We did simply 1 elution step at 50 mM EDTA (pH 8) in 20mL, which elutes the nickel ions of the Ni-NTA beads with our His-tagged proteins, instead of 2 elutions in 250mM and 500mM imidazole, because of the issues we faced for the previous purification with our proteins containing a 6xHis-tag on the N terminus and a 10xHis-tag on the C terminus.

• After the purification, we put the elution of 01b in the dialysis buffer (same as wash buffer A) overnight. This step is supposed to remove the EDTA and the nickel ions.

• We did not concentrate our proteins because the concentration measured after dialysis was enough for our next experiments.

Results We calculated the amount of the protein after purification by measuring the absorbance at 280 nm with the Nanodrop. The values were obtained based on the absorbance at 280 nm, the molar extinction coefficient and the molecular weight of the protein 01b.

Analysis The concentrations obtained are really high but this result is biased by the fact that the absorption peak at 260 nm was really high so the absorbance at 280 nm is a mix of the absorption peak at 280 nm due to the protein content and the tail of the 260 nm peak due to the DNA content. The SDS-PAGE and Western blot will enable us to evaluate if we have indeed 01b protein in our elution, but we cannot have a precise concentration for this protein.

Aim To run the SDS-PAGE of our samples from the SR protein purification, to see if we can find our protein of interest and in which tube. By the end of this experiment, we should choose the tube with the higher amount of protein, measure the purity and concentration of the protein and use it for the coating of the aerogel.

Protocol SDS-PAGE

Remarks

• We put 30 μL of samples and 10 μL of Tris-Glycine SDS sample buffer (2X).

• We loaded 5 μL of ladder, 10 μL of supernatant and flow through and 15 μL of all the other samples.

• We run the gel for 60 min at 100V.

Results

A band can be observed around 75 kDa on the elution lane. A lot of contamination can be seen in the background of each lane.

Analysis The expected size of our 01b (SR-Avitag) construct is 36.4 kDa. The band observed around 75 kDa was also present in the 01b and 03a SDS-PAGEs, all three purifications and quality controls were done the same day, so there is a possibility that it is a new contaminant protein. Since there are no other visible bands in the elution lane, our 01b construct was not purified correctly.

Doing a western blot will help us assess where the problem might be.

Aim To do a Western Blot of the gel of the 01b construct (SR-Avitag). We will use His-tag antibodies that bind the same His tag we used for purification in order to identify specifically the purified silk protein and see in which sample it is the most present.

Protocol Western Blot

Results

Analysis The bands observed around 36-39 kDa correspond to our 01b (SR+Avitag) protein. Since the bands have a much stronger intensity in the flow through lane than the elution lane, we can suppose that our proteins did not bind the beads. A way to improve the yield would be to redo a binding and purification with the flow through.

Aim To biotinylate the Avitag of the SR fusion protein, in order to allow the binding between the SR fusion protein and the silk fusion protein. To do so, we will first use the EZ-Link NHS-PEG4-Biotinylation Kit from Thermo Fisher that allows the simple and efficient biotin labeling of purified proteins. Then, since biotin is a small naturally occurring vitamin that binds with high affinity to avidin and streptavidin, we will use this interaction to link the silk and the SR protein together. To verify the binding, we will use a mass photometer by doing interferometric scattering microscopy (iSCAT)4

Protocol Biotinylation

Remarks The extent of biotin labeling depends on the size and distribution of amino groups on the protein and the amount of reagent used. Compared to reactions involving concentrated protein solutions, labeling reactions with dilute protein solutions require a greater fold molar excess of biotin reagent to achieve the same incorporation level. We therefore calculated the amount of NHS-PEG4-Biotin:

We considered here a protein concentration of 0.1 mg/mL. Indeed, after the protein purification we obtained a final concentration of 1.01 mg/mL, but since we suspect that the sample is contaminated with proteins that get purified too, we decided to consider the concentration obtained for the silk fusion protein (0.0928mg/mL). We simply divided this concentration by the extinction coefficient of the SR fusion protein (0.91) and obtained an estimation of the final concentration for the protein of 0.1mg/mL. Molecular weight of NHS-PEG4-Biotin : 589 mg/mmol.

After the biotinylation of the SR fusion protein, we just added with a 1:1 molar ratio, the Silk fusion protein (01a) and did an iSCAT to verify the binding.

Results To estimate the biotin incorporation we used a mixture of HABA and avidin that we added to the solution containing the biotinylated protein. Because of its higher affinity for avidin, biotin displaces the HABA from its interaction with avidin and the absorbance at 500nm decreases proportionately. We therefore estimated the amount of biotin present by measuring the absorbance of the HABA-avidin solution before and after the addition of the biotin-containing sample. The change in absorbance relates to the amount of biotin in the sample.

Analysis Since the elution sample of the SR fusion protein was contaminated by other proteins, we decided to do an estimation of the concentration based on the concentration of the silk fusion protein. We therefore considered a concentration of 0.1mg/mL for these experiments. At the end of the biotinylation, we measured the absorbance at 500nm of a mixture of HABA and avidin, and a mixture of HABA, avidin and the biotinylated protein (table above). With these absorbances, we calculated the number of moles of biotin per moles of proteins and obtained around 3.5 biotin molecules per protein. Since we were expecting that the avitag would be biotinylated by one biotin, we were expecting one biotin molecule per protein. This value is therefore way too high compared to the expected one. It is possible that more than one biotin molecule was added to the avitag or that biotins were added to other parts of the protein, or to other proteins.

After the biotinylation, we mixed the SR fusion protein biotinylated with the silk fusion protein. Since the silk protein has a monomer of streptavidin, the latest should interact with the biotin molecules added on the SR protein. This should allow the binding between our two proteins of interest. To verify this interaction, we performed an iSCAT. Unfortunately, as we can see on the Fig. 43, the three peaks show a predominant population of proteins around 50kDa. This size is not expected for any of the populations. Indeed, for the SR fusion protein that has been biotinylated we were expecting a peak around 35 and 40kDa, for the silk fusion protein around 75kDa and for the complex something around 110-120kDa. This can be explained by a too low concentration of our proteins compared to a too strong noise background due to a non negligible amount of contaminant proteins, which mask our proteins of interest and the complex. Otherwise, this result may indicate either that we didn’t successfully biotinylated the SR fusion protein, the complex couldn’t be formed, or that we didn’t purify our proteins, even if we can see our proteins in the Western Blot.

Conclusion Unfortunately, these results are not very conclusive, since we were not able to prove the binding between our two proteins of interest. Moreover, by lack of time we will not be able to troubleshoot this experience nor to redo it. This marks the last experiment we did in the lab for Hestia, 10 days before the wiki freeze: it was time to stop :). However, since the biotin-streptavidin is one of the strongest known non-covalent interactions5

Aim To express our 03a (mSA-GFP-CBD) construct in E.Coli BL21(DE3) and then purify the obtained protein. We will do a His-Tag purification using Promega’s MagneHis purification kit. Then we will check our protein expression and purification with a SDS-PAGE and a western blot.

Aim To do the transformation of E.Coli Competent Cells with the 03a (mSA-GFP-CBD) plasmid. We used water as negative control to model the absence of plasmid. Since the bacteria will be treated with kanamycin, we expect to observe colonies growing on the 03a transformed plate, the plasmid containing Kanamycin resistance, and the absence of colonies with the negative control, since there is no antibiotic resistance.

Protocol Bacterial transformation

Results

Analysis The negative control worked as expected (A). No plasmid were uptaken by bacteria, thus no resistance was obtained. The positive control worked also as expected (B). One can see a few colonies on the Ampicillin plate, which proves that the bacteria transformation is successful in this case. Finally, the 03a plasmid transformation worked as well (C). We can observe some colonies on the Kanamycin plate. The bacterial transformation of 03a worked, so we can pursue the process by doing colony picking in a sterile environment (with a flame).

Aim o grow the bacteria transformed with the plasmid 03a in a LB medium with Kanamycin to prepare big cultures and then to induce the transcription of the plasmid 03a (mSA-GFP-CBD) for protein expression with IPTG.

Protocol IPTG induction

Results

Our first OD₆₀₀ measurements results were close to 0.6, so for sample B we decided to do a dilution of 4:5 to obtain values in the good range (0.4-0.6). For sample B’s dilution we took 4 ml of the bacterial culture and 1 ml of medium.

Analysis Since the cell culture should have a final OD₆₀₀ < 6.0 for efficient processing, and that we obtained < 6 we can use our samples for the purification.

Aim To purify our induced 03a (mSA-GFP-CBD). To do so we will use the MagneHis Protein purification kit from Promega. The kit uses paramagnetic precharged nickel particles to link in a matter of minutes His tagged proteins from the bacterial lysate. Unbound proteins will be washed away with a specific washing buffer, and we will purify our protein of interest with specific elution buffers that contain imidazole.

Protocol MagneHis Purification

Aim To run the SDS-PAGE of our samples from the 03a (mSA-GFP-CBD) protein purification. By the end of this experiment, we should choose the tube with the higher amount of protein, measure the purity and concentration of the protein and use it for the coating of the aerogel.

Protocol SDS-PAGE

Remarks We ran two gels twice : 2 with sample A and 2 with sample B in order to do a SDS and a western blot for both the samples.

Results

Analysis For both samples, we observe exactly the same results. Moreover, no real difference in the strength of the signal can be noticed between the two different samples, which means that induction with 1mM or 2mM of IPTG doesn’t really change the result.

Since we received a plasmid with a slightly different backbone than the ordered one, we expected a molecular weight of 69.5 kDa for our original GFP protein, but with the addition of a thrombin site, we should get a molecular weight of 71kDa. In our experiment, we obtain for both samples (A and B) a lane around 50kDa, which is not the expected size. This means that we have another protein that has been purified with our kit, so that contains a his-tag or a hQ tag. Moreover, some contamination can be observed in most of the samples, especially in Elution 1 and around 15 kDa. Finally, we cannot observe our protein of interest with this experiment.

We cannot see our protein of interest in this experiment. This can be due to the fact that we haven’t loaded enough samples in the wells, so we should load 20 μL for the next experiment. To verify the presence of the protein, we will do a Western Blot which is more sensitive than the SDS Page. Indeed, by using conjugated anti-His antibodies, we should be able to detect each protein that contains a his tag. A consistent contaminant was also seen in the elution lanes around 15kDa. In this General consideration of the protocol, it is written that lysozyme used to lyse the cells will elute with the fusion protein and will produce a 12.5 kDa band on a SDS polyacrylamide gel. For future experiments, to prevent the binding of lysozyme to the MagneHis during the purification, we should include NaCl in the Binding/Wash Buffer.

Aim To run the SDS-PAGE of our samples from the 03a (mSA-GFP-CBD) protein purification. By the end of this experiment, we should choose the tube with the higher amount of protein, measure the purity and concentration of the protein and use it for the coating of the aerogel.

Protocol SDS-PAGE

Remarks We loaded 20 μL of samples (instead of 10μL) in each well, to see if by increasing the amount, we can see our protein of interest.

Results

Analysis As expected, we see more proteins in the elution lane since we loaded more samples, so the overall noise is higher. Unfortunately, we are not able to see a clear band around 70kDa, and we get similar results as the first SDS-PAGE with the same contaminant protein. We should then try another protein stain, or redo the all procedure of purification in the hope to see a band at the expected molecular weight next time.

The next step would be doing a Western Blot on the gels to see if the anti-his antibodies can recognize our protein of interest if present in very low concentration.

Aim To do a Western Blot of the gel from the sample A and B of the 03a construct (GFP control protein). We will use His-tag antibodies that bind the same His tag we used for purification in order to identify specifically the purified GFP protein and see in which sample it is the most present.

Protocol Western Blot

Results

Analysis For both samples, we observe exactly the same results. Moreover, no real difference in the strength of the signal can be noticed between the two different samples, which means that induction with 1mM or 2mM of IPTG doesn’t really change the result.

In the WesternBlot, we observe in both samples two lanes in the elution; one protein around 70kDa and one around 65kDa. Since we received a plasmid with a slightly different backbone than the ordered one, we expected a molecular weight of 69.5 kDa for our original GFP protein, but with the addition of a thrombin site, we should get a molecular weight of 71kDa. In our experiment we obtained two lanes that may correspond to both the expected size. This can be explained by the fact that we didn’t add any protease inhibitor, and maybe the added site has been cleaved out in some of the proteins. Thus we obtained both versions of the GFP protein.

Conclusion In this experiment, we noticed that we successfully purified the GFP protein since we observe it in the expected elution lane at the expected molecular weight. However, we need to understand why we observe two lanes around the expected size. Since we didn’t add any protease inhibitor, we will try to use them when redoing the experiment. Moreover we will run the proteins expressed by the plasmid we will have modified by PCR, DNA digestion and ligation to remove the added site. This experiment will allow us to confirm if this double lane is indeed two versions of the GFP.

Aim To transform competent cells with our construct, express the corresponding protein and purify it. Overall, here we want to retrieve our 03a (mSA-GFP-CBD) construct, to then use it as a control coating on the aerogel. To do so, we went to the protein facility to test a new purification protocol and new bacterial strains.

Aim To grow the BL21 bacterial strains transformed with the plasmid 03a in a TB medium with Kanamycin to prepare big cultures and then to induce the transcription with IPTG of the plasmid 03a containing GFP fused to mSA, CBD and His tag.

Protocol IPTG induction

Results

Analysis In the gel we ran to see if there is the expression of our protein, we could see the expression of the protein from the 03a plasmid. Indeed, even if we obtained smaller OD₆₀₀ values than the ones PTSP is used to obtain after the IPTG induction (OD₆₀₀ between 2 and 10), we decided to do the purification.

Aim To run the SDS-PAGE of our samples from the GFP protein purification of samples coming from BL21, to find in which tube we can find most of our protein of interest. By the end of this experiment, we should choose the tube with the higher amount of protein, measure the purity and concentration of the protein and use it for the control coating of the aerogel.

Protocol SDS-PAGE

Remarks o run the SDS-PAGE of our samples from the GFP protein purification of samples coming from BL21, to find in which tube we can find most of our protein of interest. By the end of this experiment, we should choose the tube with the higher amount of protein, measure the purity and concentration of the protein and use it for the control coating of the aerogel.

Results

Analysis In the gel, we can clearly see a band around 70kDa in each elutions sample, which is the expected size of our GFP protein. During the protein purification, we had to elute the protein with a high amount of imidazole which can damage it. Therefore, we had to think of a way to dialyse the protein to get rid of this imidazole excess, without using cellulose based material (which are the standards for this purpose). However, to check whether this protein is the expected GTP protein, we will run the elution 1 sample in a Western Blot.

Aim To do a Western Blot with the elution sample 1 to see whether the protein we see around 70kDa is our GFP protein that we should have successfully purified.

Protocol Western Blot

Results

Analysis Here we clearly see a band around 70kDa. This means that a protein has been recognized by the anti-His antibodies. It allows us to confirm that this protein is indeed our GFP protein. Moreover, we obtained a really good amount of it that we decided to flash freeze and store at -20°c to let us time to figure out how to remove the excess of imidazole.

Aim To do the transformation of BL21 E.Coli Competent Cells for protein expression with the new 03a plasmid (GFP + CBD) obtained after the Bacterial transformation

Remarks

• Here we used 25 μl of E.Coli competent cells and 2.5 μl of the new 03.a plasmid

• We didn’t have gas anymore so we couldn’t work with the flame, therefore we worked under the laminar flow hood for sterile techniques.

• We didn’t do any controls because we didn’t have enough plates and we already did many transformation controls.

• We used 200 μl of SOC medium instead of 100 since we started with more cells.

Results

Analysis The 03a plasmid transformation of BL21 competent cells worked well : we can observe colonies on the Kanamycin plates.

Aim To amplify the plasmids 03a by picking bacterial colonies from bacterial transformation made on 09/09 and to grow bacteria in bigger liquid cultures.

Protocol Colony picking

Remarks We put 10 mL of LB-Kana medium in a 15 mL falcon tube and we incubated the cultures for about 20h.

Aim To grow the BL21 competent cells transformed with the new 03a plasmid in a TB medium with Kanamycin to prepare big cultures and then to induce the transcription with IPTG of the plasmid 03a containing GFP fused to mSA, CBD and His tag.

Protocol IPTG induction

Remarks We directly did 1.3L of big cultures. So we put 1.3L of TB+Kana and we added the content of the 10mL from the colony picking.

Results

Before the IPTG induction we obtained OD₆₀₀ that were between 0.6 and 0.8, which is the required range at PTSP to pursue with the induction. Then, we measured the OD₆₀₀ after the IPTG induction, and observed small values to the ones PTSP usually gets after induction (between 2 and 10), but it's sufficient to do the purification.

Analysis In the gel we ran to see if there is the expression of our protein, we couldn’t see any relevant expression. However, since we obtained good OD₆₀₀ values, we decided to do the purification. Indeed, it can be that the GFP protein is present but in very small amounts and concentration. To check this assumption we should do the purification and run an SDS-page gel as well as a Western Blot, and see if anti-His antibodies recognize the protein of interest.

Aim To purify our induced GFP fused to mSA, CBD and His tag protein. To do so, we will use the Ni-NTA affinity beads and columns of the Protein Facility (PTPSP). This purification method is used to purify His-tagged proteins. Unbound proteins will be washed away with a specific washing buffer, and we will purify our protein of interest with specific elution buffers that contain increasing concentration of imidazole. We make all the buffers ourselves.

Protocol Protein purification PTPSP

Remark After centrifugation, the pellet wasn’t green which suggests that the proteins are not expressed. For this reason, we decided to not continue the purification, since no proteins are present.

Results

Discussion Since the pellet of the transformed cells after IPTG induction and centrifugation is not green, it means that the protein is not expressed. Thus we decided to not do the purification of this protein since it is quite sure that the protein is not present. We will have to redo the purification using the old plasmid (provided by GeneScript with the unwanted additional sites that we removed by KLD cloning) to be sure to produce proteins for the other tests we need to do for the project.

Aim To transform competent cells with our construct, express the corresponding protein and purify it. Overall, here we want to retrieve our mSA-GFP-CBD protein. We will use the plasmid 03a sent by Genscript with the added site, since we were not able to produce the proteins without the site. To do so, we went to the protein facility to redo the purification protocol from 24.08.2022.

Aim To do the transformation of E.Coli Competent Cells using BL21(DE3) strain with the 03a plasmid (mSA+GFP+CBD). We will plate the cells on the LB-agar plate containing Kanamycin, and we expect the cells that have integrated our plasmid to resist those antibiotics.

Protocol Bacterial transformation

Results

Analysis The 03a plasmid transformation worked well since we can observe colonies on the Kanamycin plates. The bacterial transformation of 03a worked for BL21(DE3) strain, so we can pursue the process by doing colony picking in a sterile environment (with a flame).

Aim To amplify the plasmids 03a by picking bacterial colonies from the bacterial transformation plate made on 17/08 (has been conserved at 4°C) and to grow bacteria in bigger liquid cultures.

Protocol Colony picking

Remarks We put 50 mL of LB-Kana medium in a 50 mL falcon tube, and the cultures were incubated for about 20h.

Aim To grow the BL21(DE3) competent cells transformed with the 03a plasmid in a TB medium with Kanamycin to prepare big cultures and then to induce the transcription with IPTG of the plasmid 03a containing GFP fused to mSA, CBD and His-tag.

Protocol IPTG induction

Remark We directly did 1L of big cultures: we put 1L of TB+Kana and we added the 50mL from the colony picking.

Results Before the IPTG induction we obtained OD₆₀₀ that were between 0.6 and 1, which is the required range at PTSP to pursue with the induction. Then, we measured the OD₆₀₀ after the IPTG induction, and observed small values to the ones PTSP usually gets after induction (between 2 and 10), but it's sufficient to do the purification.

Aim To purify our induced mSA-GFP-CBD-10xHis protein. To do so, we will use the Ni-NTA affinity beads and columns of the Protein Facility (PTPSP). This purification method is used to purify His-tagged proteins. Unbound proteins will be washed away with a specific washing buffer, and we will purify our protein of interest with specific elution buffers. We make all the buffers ourselves.

Protocol Protein purification PTPSP

Remark

• After lysis and centrifugation, the pellet turned out very green. This was unexpected since the sfGFP should have been present in the supernatant. We decided to proceed with the purification anyway.

• We diluted the total pellet into 35mL of wash buffer A.

• added 175 μl of lysozyme to better lyse the cells.

• We mixed the supernatant with the beads for 2 hours and not overnight.

• Wash 2 was done with 70mM (50mL per column) instead of 50mM (30mL) to remove more unspecific binding before the elution.

• We did simply 1 elution step at 50 mM EDTA (pH 8) in 20mL, which elutes the nickel ions of the Ni-NTA beads with our His-tagged proteins, instead of 2 elutions in 250mM and 500mM imidazole, because of the issues we faced for the previous purification with our proteins containing a 6xHis-tag on the N terminus and a 10xHis-tag on the C terminus.

• After the purification, we put the elution of 01b in the dialysis buffer (same as wash buffer A) overnight. This step is supposed to remove the EDTA and the nickel ions.

• We did not concentrate our proteins because the concentration measured after dialysis was enough for our next experiments.

Results

We calculated the amount of the protein after purification by measuring the absorbance at 280 nm with the Nanodrop:

Analysis The results obtained after lysis and centrifugation can be explained by an incomplete lysis of the cells, or a deficient expression of our 03a (mSA-GFP-CBD) construct. If it is a problem of cell lysis, then we can redo a lysis using the above cell debris pellet.

We proceeded with the 03a mSA-GFP-CBD) construct purification. In case it does not yield enough proteins, we kept the cell debris pellet and would proceed to redo a lysis and purification. After the purification, we will assess the quality of our results with SDS-PAGEs and western blots.

Aim To run the SDS-PAGE of our samples from our 03a construct purification, to see if we can find our protein of interest. By the end of this experiment, we should choose the tube with the higher amount of protein, measure the purity and concentration of the protein and use it for the coating of the aerogel.

Protocol SDS-PAGE

Remarks

• We put 30 μL of samples and 10 μL of Tris-Glycine SDS sample buffer (2X).

• We load 5 μL of ladder, 10 μL of supernatant and flow through and 15 μL of all the other samples.

• We run the gel for 60 min at 100 V.

Results

In the elution lane, two distinct bands can be observed, one around 75 kDa and one around 40 kDa. Both bands can also be observed in the wash 2 lane, albeit with a lower intensity.

Analysis The band observed in the elution lane around 75 kDa is consistent with our previous purification of 03a. We can therefore assume that it is our expected protein. The band around 40 kDa resembles the contaminant protein we encountered during previous purifications. The band observed around 75 kDa was also present in the 01b and 03a SDS-PAGEs, all three purifications and quality controls were done the same day, so there is a possibility that it is a new contaminant protein instead of. If the protein observed around 75 kDa is our expected 03a, then it should also appear in the western blot.

Aim To do a Western Blot of the gel of the 03a (mSA-GFP-CBD) construct. We will use His-tag antibodies that bind the same His tag we used for purification in order to identify specifically the purified silk protein and see in which sample it is the most present.

Protocol Western Blot

Results

Analysis Looking at the supernatant, flow-through and elution lanes, we can observe similar bands around 70-80 kDa. These are analogous to the previously obtained SDS-PAGE. The sizes are coherent with the ones obtained in our previous 03a purifications. The very large bands on top can be attributed to the usually high amount of proteins loaded in the gel.

The proteins observed in the supernatant, flow-through and elution lanes correspond by size and by anti-His antibody recognition to our protein of interest. We have therefore purified our 03a (mSA-GFP-CBD) construct and will be able to use it in future experiments. We will proceed with tests on our hydrogels, aerogels, as well as silk biofilm creation tryouts.

Aim To grow the bacteria that hasn’t been transformed, to have a control, in a LB medium to prepare big cultures and then to induce the expression of potential proteins with IPTG.

Protocol Bacterial transformation

Results

For the first OD₆₀₀ measurement, we didn’t resuspent the bacteria. We did the dilution we thought was sufficient (1:3), but by re-measuring the OD₆₀₀ we got too high values (0.723). Then we decided to do a second dilution on top of the first one (2:3) and this time we obtained OD₆₀₀ in the good range (see table).

After 3 hours of incubation with IPTG, we measured the OD₆₀₀ again. The cell culture should have a final OD₆₀₀ < 6.0 for efficient processing and purification.

Aim To run the SDS-PAGE of our samples from the purification of untransformed bacteria. The goal is to show that no proteins that are from E.Coli can be purified with the kit.

Protocol SDS-PAGE

Remarks We ran 2 samples on the same gel, since we used a 15 well gel. Our sample 1 is the sample from the untransformed bacteria that has been induced with 1mM IPTG, and our sample 2 is the sample from untransformed bacteria that has not been induced with IPTG.

Results

Analysis For both samples, we observe exactly the same results. Indeed, with or without the IPTG induction we do not see a difference between sample 1 and 2. This means that the contaminant protein (at 45kDa) as well as the lysozyme (at 15kDa) are expressed without IPTG, so they are present and expressed “naturally” in this bacterial strain; BL21(DE3)pLysS.

With this experiment we can confirm the fact that the contaminant protein that is present in each of our samples comes from the bacterial strain we are using. Maybe, the contaminant protein as well as the lysozyme comes from the same plasmid. To avoid this contamination, and to confirm furthermore this assumption, we will repeat our experiment using a different bacterial strain.

Aim To remove the plasmids’ parts added by GeneScript to our plasmids’ design. These are a thrombin site, T7 tag and an additional 6x His-Tag. To do so, we will amplify by PCR our entire plasmids except the sequences integrated by GeneScript and then we will do a KLD to ligate the resulting linearized plasmid into a new circular plasmid. We will then check if we obtained the right plasmid sequences. Indeed we will transform bacteria to amplify the plasmids we modified via PCR-KLD. We will then send to sequencing the extracted amplified new plasmids to check whether we successfully obtained the right sequence.

At the end we want to obtain the plasmids we had designed to then perform new bacterial transformations in BL21(DE3), protein expression and purification of the proteins, because the additional 6x His-Tag seems to interfere with the purification of our proteins.

Aim To remove the plasmids’ parts added by GeneScript to our plasmids’ design. To do so, we amplified our entire plasmids except the sequences integrated by GeneScript . These are a thrombin site, T7 tag and an additional 6x His-Tag. This experiment will enable us to obtain a linearized plasmid with our genes of interest in the original plasmids’ design we thought of at the beginning.

Protocol PCR reaction

Remarks

• We used the plasmid DNA from the plasmids received by GeneScript (100 ng/μl)

Aim To do the transformation of E.Coli Competent Cells NEB 5-alpha with the new plasmids obtained with KLD cloning without the added sites of GeneScript.

Protocol Bacterial transformation

Results

Results

Analysis As one can see on the Figure 1 above, no colonies were observed on all plates except on the GFP-positive-control one. It is as expected for the negative control and shows that the bacteria only by itself cannot survive on the Kanamycin plate. This result also means that the NEB 5-alpha competent E. coli cells were performant: they could uptake a plasmid containing a Kanamycin-resistance gene and survive on the Kanamycin plate. However, for the three plates plated with the cells transformed respectively with the plasmid 01a, 01b and 03a, we would expect to see some colonies growing. As none of these plates show the presence of colonies, our plasmid must have been damaged before its potential uptaking by our bacteria. This means that the PCR reaction that preceded the KLD reaction or the KLD reaction itself did not work.

As no colony has grown on the plates where we transformed the bacteria with our plasmids of interest, the PCR reaction preceding the KLD reaction or the KLD reaction itself have failed. However, it is more likely that it is the PCR reaction that has not worked since the KLD is just mixing and incubating the PCR product with a mix of specific enzymes.

We need to troubleshoot the PCR reaction that took place before the KLD reaction but also the KLD itself to find out where the issue we are facing comes from. We should check the reagents we are using and the PCR machine settings. We will do a restriction digestion analysis on an agarose gel to troubleshoot this.

Aim To analyze the PCR fragments and plasmids we obtained after PCR and KLD cloning to remove the unwanted sites added by the company GeneScript and to troubleshoot why there were no colonies grown on the bacterial transformation plates after KLD. To do so we will cut the plasmids with different restriction enzymes and we will analyze the obtained fragments sizes and the PCR fragments run on an agarose gel.

By the end of this experiment, we should determine which step of the cloning did not work (PCR amplification or ligation with the KLD enzymes mix), and if PCR worked, the size of our whole fusion proteins and whether NcoI restriction site has been replaced by PmeI site.

Protocol Restriction digestion and agarose gel electrophoresis

Results

Analysis The first observation we can make is that apart from the DNA ladder, the agarose gel is empty. This means that the PCR did not work, since even the lanes with PCR fragments of 01a, 01b and 03a are all empty, this explains why the KLD and bacterial transformation did not work: there were no amplified DNA fragments to ligate and to transform bacteria with. Of course, since the digestion reactions have been done with PCR-KLD samples, it is normal to not see any band on the gel since there are no plasmids that could be ligated. In conclusion, this first KLD cloning attempt was not successful because of the PCR, we will now troubleshoot this PCR step and try again the KLD cloning.

Aim To remove the plasmids’ parts added by GeneScript to our plasmids’ design. To do so, we amplified our entire plasmids except the sequences integrated by GeneScript . These are a thrombin site, T7 tag and an additional 6x His-Tag. This experiment will enable us to obtain a linearized plasmid with our genes of interest in the original plasmids’ design we thought of at the beginning.

Since the first trial failed, we decided to double each cloning and use different types of PCR reagents. We redid the usual protocol with our reagents once, and we also did another cloning for each of the plasmids using Marine’s lab reagents to be sure that our first failure was not due to the reagents we are using. We used a different PCR machine (Marine’s lab) as it occurred that the one we were using might be defectuous.

Protocol PCR Reaction

Protocol We used the plasmid DNA from the plasmids received by GeneScript (100 ng/μl). The same primers as for PCR reaction for KLD (trial 1) were used.

Results

Analysis In the agarose gel, we cannot see anything from the PCR done with method 1. Since we did method 1 using our reagents, it means that there is a problem with our reagents, which would explain why previous PCR we did didn’t work. However, we can clearly see bands from the PCR done with method 2 done with Marine’s lab reagents. With this method, the three plasmids seem to have been amplified correctly, although the tilted DNA bands makes it harder to interpret the size precisely and the gel could have been run longer to have a better bands separation.

The 01a plasmid, mSA-silk4X-CBD, is at the expected size around 7.5kb.

The 01b plasmid, SR-Avitag, is at the expected size of around 6kb, but we can see a second higher band which might be the template plasmid with additional tags (lane 5).

The 03a plasmid, mSA-GFP-CBD, is expected to be 7kb but here it is around 6kb (lane 6). Thus GFP is smaller than the expected size, but this is maybe due to the tilted bands on the gel.

Finally let’s note that we cannot see the last two bands of the ladder (200 and 400 bp).

Since no lane appears with our reagents, it means that the PCR didn’t work for method 1. However, it worked for method 2, and it looks like we obtained the expected size. This means that we can pursue the KLD. In any case, if transformation is successful, we will sequence the amplified plasmids and perform a restriction digestion analysis to check more precisely if the KLD cloning indeed worked and that we have the exact desired plasmid sequences.

Aim To circularize and ligate our linearized plasmids amplified through a PCR reaction. To do so, we performed a KLD reaction which allows the efficient phosphorylation, intramolecular ligation/circularization and template removal in a single 5 minute reaction step at room temperature. This experiment will enable us to obtain a circularized plasmid with our genes of interest and the original plasmid’s design we thought of at the beginning.

Protocol KLD Reaction

Aim To do the transformation of E.Coli Competent Cells NEB 5-alpha with the new plasmids obtained with KLD cloning without the added sites of GeneScript. We used water as negative control to model the absence of plasmid. We used a GFP plasmid from the summer school as a positive control. Since the bacteria will be treated with Kanamycin, we expect to observe grown colonies on the plates transformed with the plasmids 4 to 6 (PCR-KLD with Marine’s reagents lab) and on the positive control plate since the plasmid contains a gene resistant to Kanamycin. We expect the absence of colonies on the negative control plate, since there is no antibiotic resistance, and on the plates transformed with plasmids 1-3 (PCR-KLD with our reagents) since we saw that the PCR didn’t work for these samples.

Protocol Bacterial transformation

Remarks

• For samples 1, 2 and 3 we took 50 μl of NEB 5-alpha Competent E. Coli cells.

• For samples 4, 5, 6 and both controls we took 30 μl of NEB 5-alpha Competent E. Coli cells.

• For the positive control, we only managed to take 3 μl of the GFP plasmid from the summer school.

• We put the same quantity of SOC medium in all samples (950 μl).

Results

Analysis In Figure 1.G, a lot of colonies can be observed. Bacteria transformed with the GFP-summer school plasmid, which contains a Kanamycin-resistance gene, were spread on this plate. Thus, this shows that the bacterial transformation worked as expected.

In the sub-Figures 1.D, 1.E and 1.F, some colonies have grown, which is also as expected. This shows that the PCR reaction with the new reagents followed by the KLD reaction might have worked well.

On the plates shown in Figure 1.A, 1.B and 1.C no colonies have grown. We tried to transform NEB DH5-alpha competent E.Coli cells with our three plasmids (01a, 01b and 03a) that went through a PCR and a KLD reaction. The PCR reaction performed on these plasmids was using the reagents from our lab. The fact that we don’t notice any colony on the plate confirms that the PCR reaction did not work as we saw on the agarose gel.

Conclusion As we observed colonies growing on the plates where the bacteria went through the PCR reaction with the new reagents, this means that our previous PCR reactions might have not worked because of our reagents. Therefore, the bacterial transformation in NEB DH5-alpha competent E.Coli cells has succeeded and we can proceed with the colonies further to see whether our plasmids now have the sequence we designed at the beginning.

Aim To amplify the plasmid 01a by picking a bacterial colony after bacterial transformation.

Protocol Colony picking

Remarks We put 8 mL of LB-Kana medium in two 15 mL falcon tubes and we did two colony pickings so that we have two liquid cultures for further experiments. We picked from two different colonies on the same plate.

Aim To send the transformed bacteria with the new plasmids 01a, 01b and 03a (obtained after PCR and KLD) from our overnight culture for sequencing to confirm that our new plasmids were taken up by bacteria and that there is no disturbing mutation within the DNA sequence.

Protocol E.coli NightSeq

Remarks We used the bacterial overnight cultures of the 01a, 01b, 03a clones

Remarks

01a Sequencing alignment file

01b Sequencing alignment file

03a Sequencing alignment file

Analysis NanodropThe concentrations obtained are good for most of the samples: almost all are enough for the sequencing (concentration range required: 40-100 ng/μl). The purity of the samples (A260/A280 ratio) varies among samples. Globally, they are low compared to the usual value 1.8, which would demonstrate that our samples have a good DNA purity.

Sequencing

• 01a : As observed in 1.B, the N domain AS modules were cut down. We can suppose that the KLD cloning did not fully work on our 01a construct. The properties of the Silk construct were probably affected, it won’t be possible to use it for a biofilm.

• 01b As shown in 2.B and 2.C the parts added by the company were successfully removed and the PmeI restriction site successfully reinserted. These plasmids can therefore be used for further experiments. As for 2.D, it is the plasmid as received from the company, which means that we cannot use it in further experiments.

• 03a As shown in 3.B, 3.C, and 3.D, each sequenced plasmid had the parts added from the company successfully removed and the PmeI restriction site successfully reinserted. These plasmids can therefore be used for further experiments.

The KLD cloning for the 01a plasmid was unsuccessful, we have to explore other ways of reconstructing our original design. For 01b, the KLD cloning was successful for the samples 1 and 2, we will therefore use these plasmids for our future experiments. For 03a, the KLD cloning was successful for the samples 1, 2 and 3. This means that we can use all of them in our future experiments.

Aim To analyze the plasmids we obtained after the removal of the unwanted sites added by the company GeneScript. To do so we will cut the plasmids with different restriction enzymes and we will analyze the obtained fragments sizes on an agarose gel.

By the end of this experiment, we should determine the size of our whole fusion proteins (GFP, SR and Silk fusion proteins) and see if the removal of the site with PCR amplification worked. Indeed, if we observe two bands by adding SalI + PmeI it means that the removal of the site and the addition of PmeI worked. Otherwise, if we observe two bands by adding SalI + NCoI, it means that the enzyme NCoI still cuts so its restriction site is still present. If so, this means that the PCR or the KLD didn’t work and we still have the wrong plasmid.

Protocol Restriction Digestion and Agarose Gel Electrophoresis

Remarks

• Plasmid DNA used was 01a, 01b and 03a Minipreps from plasmids cloned by KLD and amplified in NEB 5-alpha

• We used Version 2 during this experiment

• We incubated the digestion reactions for 3h30 at 37°C.

• We used a 24-well comb to cast the gel since we have 20 samples.

• We have 3 different constructs (01a, 01b, and 03a) and we did 6 reactions per sample, except for 01b for which we did 5 reactions (all of them except D4). Indeed, we did not have sufficient amounts of 01b new plasmid so it was not possible to do the 6 reactions.

• We loaded 21 μl of samples mixed with loading dye.

• The gel was run at 100V for 90 min.

For all plasmid (01a, 01b, 03a) we did the following reactions:

Results

Analysis By comparing the agarose gel simulation with the gel we obtained, we first observe that overall we obtained similar patterns as the one expected. Indeed, we see that plasmids digested with NcoI behave like undigested plasmids and the one digested with PmeI have been linearized like SalI, hence proving that the KLD cloning worked and that we successfully replaced NcoI restriction site by PmeI, suggesting that we also got rid of the unwanted sites.

The linearized plasmids 01a, 01b and 03a have the expected sizes, respectively ~8000 bp (expected size: 7600 bp), ~6000 bp (expected size: 6100 bp) and 7500 bp (expected size: 7100 bp), that also correspond to the PCR fragments. However, the resolution is not high and we cannot distinguish 100 bp difference, the sequence size that we removed with KLD cloning.

The lanes 7, 14 and 22 are the samples double digested with SalI and PmeI, the excised fragment should be the size of the fusion proteins sequences we want to express and the other fragment is the backbone without a gene of interest. Unfortunately, the digested samples were not concentrated enough to have a good visibility under the gel imager, especially compared to the PCR samples that were much brighter and saturated the image. This is probably why we don’t see any excised DNA fragment in the lanes 14 and 22 for 01b and 03a, but we still see that the double digestion worked because the backbones are now around 5300 bp as expected. Luckily, for 01a in lane 7 we can guess a light band around 1500 bp that corresponds to the excise fragment of the double digestion. However, this band is expected to be at 2300 bp, indicating that the silk fusion protein sequence has not been amplified correctly by PCR and that we lost about 800 bp. This is most likely due to the repetitive sequences present within the silk sequence that might have formed secondary DNA structure that messed up the PCR amplification.

In conclusion, this restriction analysis of the KLD plasmids confirms what we had concluded from the sequencing results: the KLD cloning was successful for 01b and 03a, but it did not completely work for 01a because of the required PCR step. The absence of a part of the silk sequence in the sequencing results was not a sequencing error, the cloned 01a plasmid has indeed not been amplified correctly and we cannot use it for protein expression. We will troubleshoot the cloning of 01a by doing standard NcoI digestion and ligation, since luckily 01a sent by GeneScript has two NcoI sites flanking the unwanted sequences we want to remove.

Aim To do the transformation of E. coli Competent Cells NEB 5-alpha with the good new plasmids that obtained good sequencing results after the miniprep, to have a backup plate and transformed bacteria if we need more of the good plasmids. We will therefore transform DH5a competent cells with the miniprep samples of the constructs 01b and 03a that obtained good sequencing results, since the results for 01a are really bad. We chose the sample 2 of 01b and sample 2 of 03a from the sequencing results.

Since in theory bacteria will be transformed with good circular plasmids that contain the resistance to kanamycin, we expect to see just the colonies that have been transformed with the plasmids in the plate.

Protocol Bacterial transformation

Remarks

• We used the bacterial cultures of 01b miniprep from sample 2 and 03a miniprep from sample 2

Results

Analysis In the sub-Figures A and B above, some colonies have grown, which is as expected. This shows that the PCR reaction followed by the KLD reaction have worked well for 01b and 03a. It means that we have colonies that contain the plasmids 01b and 03a that obtained good sequencing results.

We will be able to amplify the plasmid without the aded site of the company, and do other minipreps if we need purified plasmids.

Aim To purify plasmid DNA from our liquid culture of bacteria transformed with our good 01b and 03a plasmids, verified after sequencing and obtained after PCR and KLD reactions. We want once again to obtain more purified plasmids in order to transform BL21 bacteria for protein production. We took liquid cultures from the bacterial transformations we have done with the plasmids validated in the MiniPreps 01b and 03a.

Protocol Miniprep Plasmid Purification

Remarks

• We used the bacterial cultures of 01b clone and 03a clone

• We applied the protocol of 3ml of bacterial culture as starting material and used tubes of 1.5 ml.

• We did twice the washing steps with centrifugation at maximum speed for 30 seconds.

• We did twice the steps with ERB + centrifugation and Column wash + centrifugation.

• We heated the elution buffer to 50°C before using it in the last step.

• For the NanoDrop, we pipetted 1.5 μl on the machine.

• We stored the samples at -20°C until next use.

• At the beginning, we had 10 mL of bacterial liquid culture for our plasmids 01b and 03a. So, we decided to do some Minipreps with 6 mL of bacterial culture and some with 4 mL (see which samples in the table below).

Results

Analysis The concentrations obtained are not good for most of the samples: indeed, if we wanted to send to sequencing, all the samples are not enough for the sequencing (concentration range required: 40-100 ng/μl). The purity of the samples (A260/A280 ratio) varies a lot among samples. Three ratios out of 4 are really below the usual value 1.8, which would demonstrate that our samples don’t have a good DNA purity. The sample that has a good purity ratio unfortunately has the smallest concentration in DNA. We did these minipreps in order to obtain more purified plasmid that does not contain the added site. Here we obtained those plasmids but in low amounts. For the nexts experiments in which we will reuse them, mostly transformations, we will have to adapt the amounts of the other reagents to be sure to transform bacteria with enough material.

Aim To amplify the plasmids 01b and 03a by picking bacterial colonies that gave good sequencing results (bacterial transformation of 31/08 because we forgot to pick from the plates made on 06/09).

Protocol Colony picking

Remarks

• We put 10 mL of LB-Kana medium in two 15 mL falcon tubes.

• We did two colony pickings per plasmid plate so that we have two liquid cultures for further experiments. We picked from two different colonies that gave good sequencing results on the same plate for each plasmid.

• For 03a (control sfGFP), S1 = Colony 2 and S2 = Colony 3

• For 01b (SR), S1 = Colony 2 and S2 = Colony 4

Aim To purify plasmid DNA from our colony picking liquid culture of bacteria transformed with our good 01b and 03a plasmids, verified after sequencing and obtained after PCR and KLD reactions. We want once again to obtain more purified plasmids in order to transform BL21 bacteria for protein production. We took liquid cultures from the bacterial transformations we have done with the plasmids validated in the MiniPreps 01b and 03a. Here we are redoing this miniprep for the second time since the previous one (done the 2022_09_07) gave us really bad plasmid concentration.

To do this experiment we first did a colony picking on the same colonies and put it in liquid culture overnight.

Protocol Miniprep Plasmid Purification

Remarks

• We used the bacterial cultures of 01b clone and 03a clone

• We applied the protocol on 10 ml of bacterial culture as starting material and used tubes of 1.5 ml for the rest of the experiment without increasing the amount of buffer. In this way we want to obtain more concentrated samples.

• We did twice the washing steps with centrifugation at maximum speed for 30 seconds.

• We did twice the steps with ERB + centrifugation and Column wash + centrifugation.

• We heated the elution buffer to 50°C before using it in the last step.

• For the NanoDrop, we pipetted 1.5 μl on the machine.

• We stored the samples at -20°C until next use.

Results

Analysis The concentrations obtained are finally good! Indeed we obtained for all samples a concentration higher than 100 ng/ul which we never obtained before. Moreover, the purity of the samples (A260/A280 ratio) is close to 1.8 which is the usual value. This demonstrates that our samples have a good DNA purity.

Conclusion We did these minipreps in order to obtain more purified plasmid that does not contain the added site. Here we successfully obtained those plasmids and in a pretty high amount. This enables us now to transform BL21 E.Coli strain to start the protein production in order to finally purify the protein from our designed construct.

Aim To remove the plasmids parts added by GeneScript to our 01a plasmid design of the silk fusion protein since the PCR amplification and KLD didn’t work for this construct due to the repetitive modules. The parts we want to remove here are a thrombin site, a T7 tag and an additional 6xHis-tag. To do this, we will digest our plasmid with the NcoI enzyme since there are NcoI restriction sites on both sides of the added site. Then we will run the digested plasmid on an agarose gel in order to purify the right plasmid and then we will ligate it back. Finally we will perform a bacterial transformation in NEB 5-alpha E. coli competent cells to amplify and analyze the sequence of the obtained plasmid.

At the end we want to obtain the plasmid of 01a (silk protein) we had designed to then perform new bacterial transformations in BL21(DE3), protein expression and purification of the proteins, because the additional 6xHis-tag seems to interfere with the purification of our proteins.

Aim To cut out the added site of the GeneScript company by performing a digestion of the 01a (mSA-silk(N[AS]4C)-CBD) plasmid. Since the construct contains two NcoI restriction sites on both sides of the added site, we will digest the plasmid with this corresponding enzyme. We will then run the digested product on an agarose gel to separate restriction fragments and to cut out the good plasmid, to purify and re-ligate it.

Protocol Digestion

Remarks

• 12-well comb used to cast the gel.

• We loaded two samples each with 20 μl of DNA (around 800ng DNA) per well, and 4 μl of the original plasmid .

• For the electrophoresis, we first ran at 100 V for 1 hour but samples had barely migrated so we added 50 min at 100 V.

Analysis

Analysis We can see on the 01a digested lanes of the agarose gel, a clear band around 7000 kDa as well as a small band around 100 kDa which should be the excised fragment. Since the expected size of the digested plasmid should be 7584 kDa, the bright bands should be the digested plasmid. Moreover, for the uncut plasmid of lane 2, we observe two principales bands: one around 9000kDa and one around 5500 kDa. The hupper band should be the supercoiled form of the uncut plasmid and the lower one the nicked circular plasmid DNA, which is surprisingly smaller than expected (7686 kDa).

Conclusion With this agarose gel, it looks like the digestion worked since we observe a clear band around the expected size. The next step will be to cut out these bands from the gel and to purify the obtained plasmid from it to ligate it back.

Aim To purify the digested plasmid of 01a we isolated with the agarose gel. To do so we used the promega Wizard SV Gel and PCR Clean-up System Quick kit. At the end of this experiment we should obtain a solution which contains our linearized plasmid of interest 01a that has been digested by NCo1, so that does not contain the added site of the company anymore.

Protocol PCR Clean u protocol

Remarks

• The empty eppendorf weight was 0.943g and with the gel inside it was 1.188g, so the final weigth was 0.245g. Thus we added 245 μl of membrane binding solution in step 2.

• At the end we obtain a final solution of 50 μl.

Results

Analysis Since we have a 50μl final solution and that the concentration is around 12.5, the final amount of linearized plasmid that we purified is 625 ng. Since we started with an initial plasmid amount of 800 ng before digestion, it means that we’ve lost around 20% of the plasmid with the digestion and purification experiment. This means that around 80% of the initial amount of plasmid have been successfully digested and purified, which is really good and sufficient to pursue with the ligation experiment. Moreover we obtained a good A260/280 ratio (close to the 1.8 optimal one) which means that we have a very good plasmid purity.

Aim To ligate back the digested plasmid of 01a we isolated with the agarose gel and purified with a promega purification kit. Indeed, we obtained 625ng of purified linearized plasmid at the end of the purification on gel, and for the first trial of ligation we took 170ng so there are 455ng left. For this experiment we will therefore use the 455ng that are left. To do so we used the NEB Quick ligation kit. At the end of this experiment we should obtain a solution which contains our circular plasmid of interest 01a that has been digested by NCo1, so that does not contain the added site of the company anymore.

Protocol Quick ligation

Remarks

• We didn’t do the heat denaturing step.

• Certainly due to pipetting mistakes or evaporation, we were left with 425 ng and not 455 ng of DNA.

We did just 1 sample, and multiplied by 4 all the amounts:

Discussion We will be able to see the result of this experiment after the bacterial transformation if we observe grown bacteria. If not, it would mean that there was a problem with the ligation and that the bacteria has been transformed with a linear plasmid that does not provide the kanamycin resistance.

Aim To redo the transformation of E.Coli Competent Cells NEB5-alpha with the ligation product (trial 2) that this time we do not heat inactivate since we found out that heat inactivation of Quick ligase reduces transformation efficiency. Since in theory bacteria will be transformed with good circular plasmids that contain the resistance to kanamycin, we expect to see just the colonies that have been transformed with the plasmids in the plate.

Protocol Bacterial transformation

Remarks

• We didn’t have gas anymore so we couldn’t work with the flame, therefore we worked under the laminar flow hood for sterile techniques.

• We used 50 μl of E.Coli NEB5-alpha competent cells instead of 15.

• We put 20 μl of the ligation mix (trial 2), which corresponds to ~100 ng of DNA.

• We didn’t do any controls because we didn’t have enough plates and we already did many transformation controls.

• Cells were put on ice for 30 minutes after adding the DNA, not 10 min, and for 5 minutes instead of 2 after heat shock.

• We used 200 μl of SOC medium instead of 100 since we started with more cells.

Results

Analysis Here we finally obtained colonies after the second ligation trial and the third transformation. Since we now observe colonies, it means that the plasmid has been successfully circularized, so that the ligation worked. The last step of the ligation protocol mentions to not heat inactivate the ligase since it reduces transformation efficiency. It is probably this inactivation step that was a problem for the first two trials.

Aim To amplify the plasmids 01a by picking bacterial colonies from bacterial transformation made on 09/09 and to check if the cloning worked by sending the samples (liquid cultures and Minipreps) for sequencing. We want to do a lot of colony picking to increase the chance of finding at least one colony for which the integrated plasmid is the desired cloned version, since we are not sure that we took only backbones that were double digested and lost the unwanted sites from GeneScript.

Protocol Colony picking

Remarks

• We put 10 mL of LB-Kana medium in each 15 mL falcon tube.

• We did 12 colony pickings from the transformation plate 01a DH5-alpha 09/09/22. Pickings 1 and 2 were done on 11/09/22 and 3 to 12 on 12/09/22 because colonies were very small on 11/09/22.

• We incubated the cultures for about 20h.

Aim To purify plasmid DNA from our overnight culture of bacteria transformed with our good 01a plasmid obtained after digestion and ligation experiments. We will then send them to sequencing, to confirm that our new plasmids were taken up by bacteria and that there is no mutation within the DNA sequence.

Protocol MiniPrep

Remarks

• We used the bacterial cultures of 01a clone with the right new plasmid

• We applied the protocol on 10 ml of bacterial culture as starting material and used tubes of 1.5 ml for the rest of the experiment without increasing the amount of buffer. In this way we want to obtain more concentrated samples.

• We did twice the washing steps with centrifugation at maximum speed for 30 seconds.

• We did twice the steps with ERB + centrifugation and Column wash + centrifugation.

• heated the elution buffer to 50°C before using it in the last step.

• For the NanoDrop, we pipetted 1.5 μl on the machine.

• We stored the samples at -20°C until next use.

• For the E.Coli NightSeq, we added 10 uL of our bacterial cultures to the dedicated tubes that contain the cell lysis buffer given by Microsynth.

Results

Sequencing alignment file

Analysis The concentrations obtained using Nanodrop are good for our two samples: for both, it is enough for the sequencing (concentration range required: 40-100 ng/μl). The purity of the samples (A260/A280 ratio) is also satisfying since it is around the expected value of 1.80 which shows that we have a great purity.

For the sequencing, one can see that the reading frame of our fusion protein is intact in sample 1. Same can be observed on Figure 2 for sample 2. Also, on the sequencing document containing all the alignments linked above no important mutations are detected, only one deletion at the beginning of the sample 1’s sequencing. This means that both the sequencing and the digestion-ligation followed by the bacterial transformation of plasmid 01a have been successful.

Conclusion Globally the sequencing results are good. We can work further with both cultures. We could prefer to work first with the culture of sample 2 since it has a higher plasmid’s concentration than sample 1.

Aim To purify plasmid DNA from our overnight culture of bacteria transformed with our new 01a, 01b and 03a plasmids obtained after PCR and KLD reactions. We will then send them to sequencing, to confirm that our new plasmids were taken up by bacteria and that there is no mutation within the DNA sequence.

Protocol Miniprep Plasmid Purification

Remarks

• We used the bacterial overnight cultures of 01a, 01b, 03 clones.

• We applied the protocol of 3ml of bacterial culture as starting material and used tubes of 2 ml.

• We did twice the washing steps with centrifugation at maximum speed for 30 seconds.

• We did twice the steps with ERB + centrifugation and Column wash + centrifugation.

• For the NanoDrop, we pipetted 1 μl on the machine.

• We stored the samples at -20°C until sequencing.

• We pipetted 15μl of our samples, for sequencing with primers T7 and T7 Term from their Standard Primer List.

Results All 9 samples were sent to Microsynth.

Sequencing

01a Sequencing alignment file

01b Sequencing alignment file

03a

Analysis NanodropThe concentrations obtained are good for most of the samples: almost all are enough for the sequencing (concentration range required: 40-100 ng/μl). The purity of the samples (A260/A280 ratio) varies among samples. Globally, they are low compared to the usual value 1.8, which would demonstrate that our samples have a good DNA purity.

Sequencing

• 01a : As observed in 1.B, the N domain AS modules were cut down. We can suppose that the KLD cloning did not fully work on our 01a construct. The properties of the Silk construct were probably affected, it won’t be possible to use it for a biofilm.

• 01b As shown in 2.B and 2.C the parts added by the company were successfully removed and the PmeI restriction site successfully reinserted. These plasmids can therefore be used for further experiments. As for 2.D, it is the plasmid as received from the company, which means that we cannot use it in further experiments.

• 03a As shown in 3.B, 3.C, and 3.D, each sequenced plasmid had the parts added from the company successfully removed and the PmeI restriction site successfully reinserted. These plasmids can therefore be used for further experiments.

Conclusion The KLD cloning for the 01a plasmid was unsuccessful, we have to explore other ways of reconstructing our original design. For 01b, the KLD cloning was successful for the samples 1 and 2, we will therefore use these plasmids for our future experiments. For 03a, the KLD cloning was successful for the samples 1, 2 and 3. This means that we can use all of them in our future experiments.

Aim To purify plasmid DNA from our culture of bacteria transformed with our new 01a, 01b and 03a plasmids obtained after PCR and KLD reactions once again to obtain more purified plasmid. We choose samples from the culture that obtained good results in the first sequencing.

Protocol Miniprep Plasmid Purification

Remarks

• We used the bacterial cultures of 01a clone from sample 3, 01b clone from sample 2 and 03a clone from sample 2 and 3

• We applied the protocol of 3ml of bacterial culture as starting material and used tubes of 1.5 ml.

• We did twice the washing steps with centrifugation at maximum speed for 30 seconds.

• We did twice the steps with ERB + centrifugation and Column wash + centrifugation.

• For the NanoDrop, we pipetted 1 μl on the machine.

• We stored the samples at -20°C until next use.

• For samples 01b 2.2 and sample 03a 2.2 we started just with 1.5 mL of culture since we didn't have enough culture to do 2 different samples.

Results

Analysis The concentrations obtained are not so good for most of the samples: indeed, if we wanted to send to sequencing, almost all the samples are not enough for the sequencing (concentration range required: 40-100 ng/μl). The purity of the samples (A260/A280 ratio) do not vary so much among samples. Globally, they are around the usual value 1.8, which would demonstrate that our samples have a good DNA purity.

We did this miniprep in order to obtain more purified plasmid that does not contain the added site. Here we obtained those plasmids but in relatively low amounts. For the nexts experiments in which we will reuse them, mostly transformations, we will have to adapt the amounts of the other reagents to be sure to transform bacteria with enough material.

Aim To test the binding of our Cellulose Binding Domain to cellulose. To do so, we will use a nitrocellulose membrane (for western blot) and we will add our mSA-GFP-CBD protein. To compare the affinity to this membrane, we will do the same experiment with a GFP that does not contain CBD. We will therefore do several washes using PBS to wash out the proteins that are not bound to the membrane. By the end of this experiment we expect that our mSA-GFP-CBD protein binds more to cellulose than the GFP without CBD.

Protocol Drop-Plot

Remark We used the mSA-GFP-CBD (03a) protein purified with a final concentration of 0.44ng/ul, and the GFP (control) protein purified re-diluted in the wash Buffer A (300mM NaCl + 20mM Hepes) to have a concentration of 0.44ng/ul

Results

Analysis Even before the first washing step, we could see under the UV light that our GFP protein is more fluorescent than the control GFP. This is a bit surprising since both GFP have the same concentration. In this way, after the first washing step we could clearly see that our GFP protein was still more fluorescent than the control GFP ((fig 2.A)). Moreover, by comparing the first washing step with the third one ((fig 2.A) and (fig 2.B)), we observed a diminution in the intensity of the fluorescence of the control GFP, while it is not the case for our mSA-GFP-CBD. With these observations we can conclude that in this experiment, our protein mSA-GFP-CBD looks to have a higher affinity with the nitrocellulose membrane than the GFP control protein. However, to better characterize this observation, we should redo the experiment, analyze and quantify the fluorescence using special software as ImageJ.

Analysis Even if by eye it looks like our mSA-GFP-CBD protein binds more the nitrocellulose membrane than the control GFP, we should redo this experiment with better quantification of the fluorescence using a microscope for the pictures and the ImageJ software for the measurements. Also, we would like to perform some pH washes (where we will increase progressively the pH) to see to which extent our CBD domain binds to cellulose.

Aim The aim of this experiment is to test the binding of our Cellulose Binding Domain to cellulose. To do so, we will use a nitrocellulose membrane (usually used for western blot) and we will add our mSA-GFP-CBD protein. We will establish a control by doing the same experiment with a GFP protein that does not have a CBD domain. We will do three washes with PBS to wash out the proteins that are not bound to the membrane. Then, we will proceed to pH washes where we will decrease the pH to see to which extent the CBD domain binds cellulose. By the end of this experiment we expect that our mSA-GFP-CBD protein binds more to cellulose than the GFP without CBD.

Protocol Drop-Plot

Remark We used the mSA-GFP-CBD (03a) protein purified with a final concentration of 0.44ng/ul, and the GFP (control) protein purified re-diluted in the wash Buffer A (300mM NaCl + 20mM Hepes) to have a concentration of 0.44ng/ul

Remarks

• We used the mSA-GFP-CBD (03a) protein purified with a final concentration of 0.44ng/ul, and the GFP (control) protein purified re-diluted in the wash Buffer A (300mM NaCl + 20mM Hepes) to have a concentration of 0.44ng/ul

• We used a HCl stock (0.01M) to do several washes:

• Wash 1 : pH5 → 10^-5 Mol = 10 uL wash 2 + 990 uL water

• Wash 2 : pH3 → 10^-3 Mol = 10 uL wash 3 + 990 uL water

• Wash 3 : pH1 → 10^-1 Mol = 900 uL HCl + 100 uL water

Results

Analysis One can see on (fig 1.B), (fig 1.C) and (fig 1.D) that the PBS washes did not decrease the fluorescence of both our recombinant protein (mSA-GFP-CBD) and the control (GFP without a CBD domain). That means that it did not affect the bonding of both proteins to the membrane. Then, on (fig 1.E) we can observe that the fluorescence did not change at all the same way as with the PBS washes. Then, a solution of pH5 is not acid enough to break the bond between the proteins and the membrane. However, on (fig 1.F), one can see that the fluorescence totally disappeared for both proteins on the membrane. Thus, a solution of pH3 is acid enough to break the bond between the proteins and the membrane and to wash them out.

We now know that the bond between both our protein of interest (mSA-GFP-CBD) and our control (GFP without a CBD domain) can be broken when they are exposed to an acidic solution of pH3. The next step would be to do smaller pH-steps in the pH-washes (ideally of 0.5) to determine how fine the CBD domain binds to cellulose in comparison to an usual protein that does not have any CBD domain. The results we would like to get is to still observe fluorescence for our recombinant protein (mSA-GFP-CBD) and no more for our control (GFP without a CBD domain).

Aim To see if our construct 03a (mSA-GFP-CBD) binds to the hydrogel, to prove that the CBD domain we added to our construct allows the linking with the aerogel. We will use as positive control a GFP that doesn’t contain the CBD domain and double-distilled water as negative control. We will send all soaked hydrogels to the freeze drier to produce an aerogel. If the aerogel turns green when it is exposed to UV, it would mean that our GFP proteins are not denatured with the freeze-drying, that they remain on the aerogel and that they effectively bind the hydrogel.

Protocol Hydrogel coating

Remarks

• We used mSA-GFP-CBD (03a) protein purified with a final concentration of 0.44ng/ul and GFP (control) protein purified re-diluted in the wash Buffer A (300mM NaCl + 20mM Hepes) to have a concentration of 0.44ng/ul

• In our case, we had a hydrogel of 1.35 cm^3 (= 1.35 ml) into a petri dish of 7.7 cm^3 (=7.7 ml). Moreover, the height of the hydrogel was 3mm, so by adding a volume of 1.5ml it should be enough to soak the hydrogel entirely in the petri dish.

• Here we used 3 hydrogel and we added 1.5ml of water on the first one, 1.5ml of control GFP (without CBD) at a concentration of 0.44ng/ul on the second one, and 1.5ml of mSA-GFP-CBD at a concentration of 0.44ng/ul on the third one.

Results

Analysis Right after the first soaking step, we cannot differentiate the samples under the UV light (fig 2.A). The GFP and mSA-GFP-CBD hydrogel look a little green, but it is not very stringent. After the second soaking step, we observed a remarkable fluorescence of the mSA-GFP-CBD hydrogel which surprised us (fig 2.B). Indeed, we expected to see the same fluorescence between the GFP and the mSA-GFP-CBD samples. This result suggests that our protein looks to bind better the hydrogel than the GFP control, which is what we want. Finally, after the removal of the proteins, we cannot see a difference between the hydrogels (fig 2.C). It is possible that the proteins are absorbed by the hydrogel. To check this hypothesis, we proceeded to the freeze drying step. As our aerogel soaked in mSA-GFP-CBD show some fluorescence under UV light, it is therefore possible that our protein of interest is bound to the aerogel.

Conclusion Final freeze-dried aerogels showed some fluorescence under the UV light. Therefore, the freeze-drying step does not denature our proteins and we managed to attach our protein of interest to the aerogel. We could redo this experiment and take clearer images with a fluorescence microscope. In addition to that, we would like to better quantify the fluorescence of each final aerogel after freeze-drying to prove in depth the attachment of our mSA-GFP-CBD to the cellulose matrix.

Aim To see if our construct 03a (mSA-GFP-CBD) binds to the hydrogel, to prove that the CBD domain we added to our construct allows the linking with the aerogel. We will use as positive control a GFP that doesn’t contain the CBD domain and double-distilled water as negative control. We will send all soaked hydrogels to the freeze drier to produce an aerogel. If the aerogel turns green when it is exposed to UV, it would mean that our GFP proteins are not denatured with the freeze-drying, that they remain on the aerogel and that they effectively bind the hydrogel. We decided to redo this experiment to take some images with a fluorescence microscope for the hydrogels’ soakings and a fluorescence gel imager for the freeze-dried aerogels.

Protocol Hydrogel coating

Remarks

• We used mSA-GFP-CBD (03a) protein purified with a final concentration of 0.44ng/ul and GFP (control) protein purified re-diluted in the wash Buffer A (300mM NaCl + 20mM Hepes) to have a concentration of 0.44ng/ul.

• In our case, we had a hydrogel of 1.35 cm^3 (= 1.35 ml) into a petri dish of 7.7 cm³ (=7.7 ml). Moreover, the height of the hydrogel was 3mm, so by adding a volume of 1.5ml it should be enough to soak the hydrogel entirely in the petri dish.

• Here we used 3 hydrogel and we added 1.5ml of water on the first one, 1.5ml of control GFP (without CBD) at a concentration of 0.44ng/ul on the second one, and 1.5ml of GFP-CBD at a concentration of 0.44ng/ul on the third one.

Results

Analysis On (fig 81.A), (fig 81.B) and (fig 81.C), one can see that we don’t observe any fluorescence from the hydrogels soaked with double-distilled water (negative control) or GFP (positive control). Regarding the hydrogel soaked with mSA-GFP-CBD (our protein of interest), we don’t observe any fluorescence after waiting approximately 15 minutes after the first soak. However, immediately after the second soak, we could observe our mSA-GFP-CBD in solution surrounding the hydrogel (light fluorescence seen on (fig 81.B) on the right). But after waiting approximately 15 minutes, we don’t observe fluorescence anymore. These results encourage us to think that the hydrogel absorbs the proteins with different speeds depending on whether they have a CBD domain.

On (fig 82.A), one can see that we can observe fluorescence from the three aerogels produced from the hydrogels soaked respectively with double-distilled water (negative control), GFP (positive control) and mSA-GFP-CBD (our protein of interest). The aerogel with mSA-GFP-CBD shows the most intense fluorescence. It shows that after the freeze-drying step, our mSA-GFP-CBD binds more to cellulose than usual GFP. Also, we can observe some fluorescence from the aerogel with water, which might signify that cellulose shows naturally some fluorescence after the freeze-drying step. If we compare the fluorescence intensity between (fig 82.A) and (fig 82.B), it has decreased with time. However, our mSA-GFP-CBD still shows the most intense fluorescence.

Conclusion As we observe the most intense fluorescence on the aerogel with our mSA-GFP-CBD after the freeze-drying step, this shows that our mSA-GFP-CBD has bound tighter with cellulose thanks to its CBD domain compared to the usual GFP. Regarding the loss of fluorescence with time on the aerogels, we could optimize the storage of our final aerogels to avoid the loss of proteins on the aerogel. The next step could be to analyze further our fluorescence images to quantify more precisely the fluorescence, maybe by using ImageJ.

Aim To determine if by coating the hydrogel with the silk fusion protein, and then freeze drying it to make an aerogel, the latest will be more hydrophobic than without the recombinant protein. Since we already proved the efficiency of the CBD domain using the mSA-GFP-CBD (03a) protein by fluorescence quantification, we will use this conclusion and coat hydrogels with the mSA-silk-CBD (01a) construct. After the freeze drying of the hydrogel to produce aerogel, we will apply different droplets of water on top of each of them, and we will quantify the time it takes for the water to be absorbed. We expect that the aerogel with silk will have longer delays than the other aerogels. This will prove that the CBD domain we added to our silk construct allows the linking with the aerogel and that this protein is a protective coating for the aerogel.

We will use as positive control a GFP that doesn’t contain the CBD domain and double-distilled water as negative control. Moreover, we will do the experiment also with the mSA-GFP-CBD (03a) protein, to hopefully show that not any protein can increase the hydrophobicity of the aerogel, or at least not as much as the silk.

Protocol Hydrogel coating

Remarks

• We used mSA-GFP-CBD (03a) protein purified with a final concentration of 0.0942 ng/μL (1.319 μM), mSA-Silk-CBD (01a) protein purified with a final concentration of 0.0928 ng/μL (1.193 μM) and GFP (control) protein kindly purified and provided by the PTPSP, that we re-diluted in the wash Buffer A (300mM NaCl + 20mM Hepes) to have the same amount of moles in 600 μL as the number of moles soaked for 03a and 01a (7.64 x 10-10 mol).

• We used the algorithme created by the modeling team to calculate the exact amount of proteins to add on top of the hydrogel so that proteins would populate the entire surface of the aerogel. For the chosen parameters of 30 mm diameter and 3 mm thickness of aerogel, we found that approximately we could coat our aerogel with 600 uL of proteins since the experimental protein concentrations were really close for both 01a and 03a (around 1.2 μM).

Results

Analysis In this experiment, we first coated hydrogels with proteins (either the silk fusion protein (01a), the GFP fusion protein (03a), the GFP control protein or water) (fig 1.A). Then we sent them to freeze dry (fig 1.B). After this step, we saw that the coated aerogels had a more homogeneous shape, with less cracks and a sort of protective film on top of it (fig 2.B) compared to the uncoated ones (fig 2.A). Using the Keyence microscope, we filmed the application and the absorption of 4 to 5 drops of water on our coated aerogels. Then we measured the delay time it takes for the water to be absorbed, and computed the mean for each aerogel (table).

With this data set, we performed a one-way ANOVA test to see if there is a significant difference between the means between the groups. We obtained a final p-value of 0.000576 (fig 3.A). As the p-value is less than the significance level 0.05, we can conclude that there are significant differences between the groups. However, this result does not indicate which pairs of groups are different. We therefore performed a multiple pairwise-comparison to determine if the mean difference between specific pairs of groups are statistically significant.

As the ANOVA test was significant, we could compute Tukey HSD for performing multiple pairwise-comparisons between the means of groups (fig 3.B). For each comparison with the silk fusion protein (01a), we obtained a p-value way smaller than 0.05, which means that there is a significant difference between the silk fusion protein and the other groups, while there is no significant difference among the other groups (dH20, GFP and GFP fusion protein 03a). Therefore we can conclude that aerogels absorb water more slowly when they are coated with the silk fusion protein. Indeed, in the box plot we plotted to present the different populations (fig 4), it is clear that in the case of the silk fusion protein (01a), the delay times are higher which means that the drop of water takes longer to be fully absorbed by the aerogel, suggesting that the silk coating provided some hydrophobicity to the aerogel.

Overall, these results are very promising. We could improve the experiment by adding more proteins to have a better coating, even though we added twice the amount of proteins required to cover the aerogel as a monolayer. Finally, to completely prove that the silk fusion protein is a protective coating for the aerogel, we could coat the aerogel with the silk fusion protein biofilm, since we proved that the latest is hydrophobic. Unfortunately, we were not able to do this because until now the silk biofilms we produced were sticking to the petri dish, therefore we could not displace them on top of aerogels.

Batch 1

Aim The aim of this experiment is to produce a cellulose hydrogel by dissolving the cellulose in a Thiourea:NaOH:H₂O solution (5:10:85 weight percentage) and afterwards thawing it and obtaining a hydrogel.

Protocol Critical Point Drying

Comments

• The experiment was conducted only until the hydrogel formation step of the protocol

• 100g of dispersing solution was prepared for 5g of cellulose powder, and the resulting solution was divided into two petri dishes of 10cm diameter.

• This weight ratio of cellulose powder to dispersing solution was considered as “1X”, and any change in the concentration of cellulose is to be labelled in reference to this ratio. For example, 2.5g of cellulose for 100g of dispersing solution would be 0.5X

• Rinsing was not possible due to the fragile nature of the hydrogel.

Results

After waiting 24 hours and letting the solution freeze, the hydrogel is only partially gelated. This means that some parts of the gel are gelated while the rest is still liquid.

Analysis After we washed the hydrogels, we observed that they were too liquid for the production of an aerogel and that the structure wasn’t viable. This result might be due to the fact that the concentration of cellulose wasn’t high enough to produce a structure that will trap the water, or that the freezing time wasn’t enough.

Aim Commence the solvent exchange for one of the two samples obtained from the hydrogel production. Also test the solidification of the other hydrogel sample with an additional freezing time of 24 hours.

Protocol Critical Point Drying

Comments

• The experiment was carried out from the hydrogel formation step to the ethanol solvent exchange step.

Results

After the two experiments, we see that the hydrogel submerged in ethanol had solidified. However, the solution was not solid enough to be considered as a viable sample nevertheless. In contrast, refreezing the sample had no effect on the solidification.

Aim Conduct the solvent exchange (from water to ethanol) for a 2X cellulose hydrogel sample, and consequently obtaining an alcogel.

Protocol Critical Point Drying

Comments

• 50g of dispersing solution was prepared for 5g of cellulose (for 2X).

• A 10 cm diameter petri dish was used as a mould

• The protocol was followed until the solvent exchange step

Results

As can be seen from Figures A,B and C, the 2X hydrogel sample successively went through a colour change, from yellow to orange to bright red. As it did so it also gradually solidified. A negligible shrinkage in the volume was observed, a diameter shrinkage from 8.4 cm in Figure A to 7.5 cm in Figure C approximately. Note that the initial shrinkage from 10cm to 8.4cm takes place during the initial gelation of the cellulose solution during thawing.

Analysis The colour change was expected and confirms the successful exchange of water with ethanol in the hydrogel. It has been observed in literature for organic compound aerogels to turn red during solvent exchange. The gradual solidification and volume shrinkage also confirms the solvent exchange, as ethanol doesn’t occupy the same volume as the amount of water it replaces. This phenomenon is also common in case of 100% ethanol baths.

This experiment was a success as we produced an alcogel through solvent exchange. The fact that the solvent exchange induces a gradual solidification is also useful in handling the alcogel, as the hydrogel is usually fragile and requires additional caution. The sample can proceed to the critical point drying step.

Aim Establishing a concentration gradient and proceeding until alcogel production. Determining if the cellulose concentration and the solvent exchange interact with each other on the structural stability of the gel, and which of the two has a bigger effect on it.

Protocol Critical Point Drying

Comments

• Six samples on a concentration gradient were prepared, all with a 50g dispersing solution. The aimed concentrations and the corresponding cellulose weights are indicated below:

Table 47Weight required for a certain concentration

Weight of Cellulose [g]	Concentration
3.75	1.5X
4.375	1.75X
5.625	2.25X
6.25	2.5X
6.875	2.75X
7.5	3X

• Standard 10cm diameter petri dishes were used as moulds

• The protocol was carried out until the ethanol solvent exchange step

Results

All the gels have acquired an orange-red colour and hardened, with the colour getting slightly darker as the concentration of cellulose gets higher. For lower concentrations (1.5X and 1.75X), it is possible to see outlying flakes and layers of cellulose on the gel. Similar shrinkages in diameter were observed in all gels:

Analysis The colour change into red indicates that the solvent exchange was successful, a fact also supported by shrinkage in the diameter. The hardening of the gels seemed to be uniform, with no distinguishable difference in consistency of the solid structures of the alcogel being observed, yet the effect of the cellulose concentration gradient seems to be visible in the colour. It seems the effect of the solvent exchange with ethanol on hardening is considerably higher than increased cellulose concentration. Lastly, the outlying layers of cellulose are probably due to sections of the solution that stuck on the walls of the petri dish having thawed and polymerised, and then having come off and fallen onto the surface of the gel.

Batch 4

Aim Producing two samples of 15cm diameter cellulose aerogels. One of the samples would proceed with the solvent exchange and critical point drying (CPD) and the other sample would proceed with lyophilisation/freeze-drying.

Protocol Critical Point Drying

Comments

• The usage of 15cm diameter moulds required an upscaling of the hydrogel recipe. This was done by multiplying the total amount of the recipe by the ratio of the base area of a 15cm diameter petri dish to the base area of a 10cm diameter petri dish, which turned out to be 2.25. Meaning, the total dispersing solution weight in each sample was 112.5g.

• 1X → 5.625g of cellulose, 1.25X → 6.328g of cellulose

• It was decided that 1X would proceed with lyophilisation, while 1.25X would go through solvent exchange and CPD dried. This notebook will follow the 1.25X and the CPD process

Results

The hydrogels successfully gellified. The 1.25X sample also shrank in diameter and turned orange-red after the solvent exchange.

Analysis The gelification being successful, and the solvent exchange being confirmed with shrinkage and colour change, the samples were deemed ready for freeze-drying and CPD respectively.

Aim Producing a cellulose aerogel from the cellulose alcogel with a critical point dryer.

Protocol Critical Point Drying

Comments

Critical point drying is a process where the ethanol is purged from the environment and the sample and is replaced with liquid CO₂. Afterwards the supercritical point of CO₂ is reached and the resulting supercritical liquid induces no surface tension to the pores of the cellulose structure. The CO₂ is then converted into gas, resulting in the formation of the aerogel. If a normal phase change was to be the case, the surface tension and the capillary forces would make the pores collapse.

Results

The colour of the resulting aerogel was white, and the cracks from the hydrogel persisted. Little crumbs in the chamber were observed. The texture of the material also featured a mostly flat yet a little hilly composition.

Aim To produce 3 aerogel samples in cylindrical shape of 80ml in volume.

Protocol Freeze Drying

Comments

• Three samples were made with different concentrations; 0.75X, 1X and 1.25X respectively.

• Three dispersing solutions were prepared, each 80g in weight, with the same weight proportions as the original protocol. 100ml beakers were used as moulds.

Results

Analysis The cracks, deep or shallow, big or small, present on the aerogel samples are mostly due to the cracks originally appearing in the thawing and gelation process. In some cases, the cellulose sticks to the mould during freeze drying, resulting in cracks in the overall structure as well.

The post freeze-drying discoloration is most probably due to the presence of the Thiourea and NaOH salts still present in the sample, not effectively removed.

Ultimately this experiment shows that more 3D structures require additional care in handling the sample during the hydrogel phase, and exploring ways to remove the salts more efficiently. The consistency of the texture seems to be linked to an increased cellulose concentration, but the cracks are independent of that and are linked to the gentleness in the handling of the samples.

AimTo produce 3 aerogel samples in cylindrical shape of 40ml in volume, while understanding the possible causes of the post freeze-drying reddening and structure retention.

Protocol Freeze Drying

Comments

• Three samples were made with different concentrations; 0.75X, 1X and 1.25X respectively.

• Three dispersing solutions were prepared, each 50g in weight, with the same weight proportions as the original protocol.

• 50ml beakers were used as moulds.

• The 1X sample was freeze-dried for an hour less.

• 0.75X was not thawed before the freeze-drying process.

Results

Analysis The thawing and the gelation process was confirmed to be crucial, as without this process the freeze dried 0.75X sample didn’t have any consistent 3D structure at all, and presented a significant adhesion to the beaker wall. The shrinkage in volume was expected and was consistent with the prior experiments.

The considerable and relatively fast reddening of the 1X sample after freeze drying shows that the reddening is equally connected to the presence of water in the sample. Adequate freeze drying to remove any remaining water presence in a sample is therefore crucial. Silica beads to absorb the humidity for storage purposes is recommended.

The 1X gel, ignoring the discoloration, had an excellent texture and a consistent structure. This shows that proper and gentle handling of the samples after gelation can yield significantly better texture with minimal cracks/cavities.

AimProducing three samples of cellulose aerogel with a concentration gradient of 0.5X - 0.75X - 1X. Observing the possible causes of cracks and crumbs from the aerogel along the way. A dispersing solution with Thiourea, Sodium Hydroxide and Distilled Water (5:10:85) is prepared to dissolve the cellulose powder.

Protocol Freeze Drying

Comments

• Making sure that the samples are completely frozen as they are put into the freeze dryer is crucial (0.75X sample was thawed to show this).

• The hydrogels, especially at low concentrations of cellulose, are extremely fragile. As such minimal disruption of the hydrogels should be the case.

• Homogeneous dispersion of the cellulose is equally important, as such one must stir for as long as it takes.

• For clarity, 1X concentration is for 5g of cellulose powder per 100g of dispersing solution (5:100 weight ratio)

Results

Analysis The 0.5X sample cracking was unexpected, as the procedure was followed without any problems. This might be due to excessive drying, not enough cellulose to sustain the overall structure of the gel, or simply by the fact that the hydrogel stuck to the petri dish while drying and couldn’t contract as it should, thus resulting in breaking.

The 0.75X shattering was expected, as the sample was put into the freeze dryer while not being completely frozen. The liquid water most probably boiled and shattered the hydrogel. The crack line most probably formed during

The 1X sample featuring small cracks was expected.

AimProducing an aerogel sample in a 50ml tube mould and another sample in a 15cm diameter petri dish mould. Observing the reddening and texture consistency.

Protocol Freeze Drying

Comments

• Both samples were prepared with a 1.5X concentration of cellulose, as previous experiments showed this concentration to be the one that gives the best texture for the minimal amount of cellulose.

• 125g of dispersing solution was prepared for the 15cm diameter petri dish sample, while a 50g dispersing solution was prepared for the 50ml tube.

• The gradual colour change of the cylindrical tube sample was observed over the course of four days.

Results

Analysis The discoloration of the tube sample in the form of bands was probably due to the fact that the sample was freeze dried in a vertical position, with the tip pointing down and the bottom/base pointing up. This conformation possibly led to uneven drying, with the bottom completely drying and the tip and the centre not drying fully. The more prominent red colour at the centre might be due to the fact that the centre of the samples retained more water than the other parts, where the water would be the last to leave through the surface during lyophilisation. The discoloration of the petri dish sample, in contrast, was more uniform and even, as it was dried in a horizontal confirmation with maximum surface area facing the vacuum.

The texture of both samples are consistent, featuring either no cracks or superficial cracks. No significant adhesion to the mould wall was observed, also supporting the excellent texture. This tells us that 1.5X concentration gives the best structural and textural consistency for minimal amount of cellulose among all the concentrations we have tried.

AimProducing 1.5X cellulose aerogel samples for GFP-CBD attachment tests.

Protocol Freeze Drying

Comments

• Two types of samples, both having a 1.5X cellulose concentration, of different shapes were produced.

• The first type was produced after a 50ml beaker as a mould, with a cylindrical shape.

• The second type was small circles obtained by slicing a hydrogel which was produced with a 50ml tube as a mould (long cylindrical shape).

Results

Analysis The production process worked as desired. All the samples were dried appropriately, the texture that was obtained was uniform without any cracks, indicating a successful gelation and an appropriately gentle handling of the sample. Only a slight discoloration after freeze-drying was observed, which didn’t progress further, most probably due to humidity in the air than any remaining water in the sample.

This shows that the 1.5X concentration is the ideal concentration for structural consistency and optimal texture indeed.

AimProducing a concentration gradient of cellulose aerogels for the compression tests.

Protocol Freeze Drying

Comments

• Three aerogel samples were made with 1X, 1.5X and 1.75X concentration respectively.

• 50ml tubes were used as moulds, and the resulting cylindrical aerogels were then cut into even surfaced pieces for the compression test with a surgical knife.

Results

Analysis The production process worked mostly as desired. All the aerogels have a stable structure, yet have textural differences. This was expected as it is in agreement with the previous experiments. The difference in discoloration might be due to not enough freeze-drying, though this is hard to determine as the samples are usually pure white at the end of the freeze-drying process without necessarily displaying a visible indication of humidity.

For the results of the compression test, please see the Results Page.

AimProducing Aerogels using solvent exchange to extract the gelation salts.

Protocol Freeze Drying

Comments

The following implementation was considered after the discussion with EMPA’s material science experts. They suggested exploring the solvent exchange to get better aerogel results. The team went through some documentation using solvent exchange for aerogels6. In the experiments, we conducted different tests using bi-distilled water and 70 % ethanol. The solvent exchange steps consist of bathing the hydrogels into another solvent so that the liquid in the batch takes the place of the original solvent containing salts. It was crucial to execute successive baths to ensure proper exchange of liquid.

• The samples in (fig.56), had all of them a volume of 42,5 g of water, 5 g of NaOH and 2.5 g of Thioria. Their concentration of cellulose was different for (fig.56 A,C) the initial cellulose concentration was 3.75 g (1,5x Thioria content) and for (fig.56, B), the initial cellulose concentration was 1,875 g ( 0.75 x Thioria content). Here we produced three samples.

• The samples in (fig.57 ) had all the same concentration of cellulose compared to Thioria, which was 0.75 times. Those were different volumes (fig57. A,C) were cut from a 50ml tube hydrogel and (fig57. B,D) were cut from a 14ml tube. Here we considered four samples.

Results

Analysis The aerogel samples in (fig56. A) and (fig 56. B), had a solid structure compared to the aerogel sample in (fig56. C). The fact of using only bi-distilled water solvent exchange during two baths diluted the structure of the hydrogels during the dilution of the salts. The 1.5x solvent exchanged sample (fig56. C) weighed 9.795 g, compared to 14.430 g for the sample (fig56. A) without the solvent exchange step. We concluded that using only bi-distilled water fragilized the linkage between cellulose molecules and that we should explore other solvents to insure keeping the structure of our aerogels.

The next experiments (fig57) showed promising results, since in (fig57. A,B) the samples seemed to keep their firm structure and didn’t turn pink after some days of storing them in an ambient temperature. The difference between the aerogels in (fig56 A) and (fig56 B) was placing the first sample in a bi-distilled water bath then ethanol at 70 % and placing the second sample directly in ethanol at 70 %. The goal of our experience was achieved by finding that the best method to execute the solvent exchange was bathing the hydrogels into ethanol at 70 % then into bi-distilled water.