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iGEM Freiburg 2022 presents
chAMBER
Design, build, test and learn.
Much of biology is concerned with the study of phenomena and their rational explanation. Synthetic biology, however, uses these phenomena to develop new applications. Engineering and tweaking of these applications are an important part of synthetic biology. Therefore, it is sometimes better to look at the problems not as scientists but as engineers.
In the engineering cycle, you first design and model your application with the intended function in mind. Second, you build and implement the proposed construct in the designated system. Third, you perform tests and measurements regarding the effect of the construct. Fourth, you analyse and quantify your data to learn whether your application has the expected result. Finally, you return to the first step and apply your knowledge to improve the system. Repeat this DBTL cycle until you are satisfied with the performance of your application.
chAMBER is a project that tackles biosynthesis via compartmentalisation. However, it comprises a great scope of subprojects, which each underwent the DBTL cycle. We will present an iteration of the cycle dealing with the engineering and the expression of the indigo pathway and the synthetic carboxysome, the wiffleball.
Design
The journey through this cycle began with the intention of proving that compartmentalising enzymatic pathways improves their yield. Therefore, we looked for different pathways to test in said compartments. After discussing several according to their complexity and the detectability of the product, we decided on a pathway to synthesise indigo, among others. Once we settled on the pathway published by Yin et al. [1], we codon-optimised variants of the required enzymes. We used different localisation systems to recruit the enzymes of interest into the compartments. By cloning the enzyme genes into a special pCDF –DUET-1 vector that allows the expression of several enzymes, four genes (TnaA, Fre, XiaI and TnaB) were incorporated into the two multiple cloning sites. Once in the backbone, the catcher sequences, a part of the recruitment tag, were added to TnaA and XiaI. The counterpart, simply “tags”, are added to the compartmentalisation proteins and enable the emerging enzymes to be recruited into the wiffleballs. If you want to learn more about each enzyme's exact design procedure and function, click here to get to our description page!
Build
We built our compartmentalisation system from several components. As mentioned above, a plasmid was engineered expressing four relevant enzymes tagged with a catcher sequence. The sequences for the single enzymes were ordered from IDT and cloned into the backbone. A very important second component of chAMBER is the compartmentalisation system, which in this case was the carboxysome, or wiffleballs, which we received as plasmid from the Kerfeld group [2]. The wiffleballs were tagged with the analogue to the catcher sequences, and both plasmids were co-transformed into competent BL21(DE3) E. coli to synthesise Indigo and Indirubin.
Test
Subsequently, we tested the yield indigo from our transformed E. coli BL21(DE3) containing the following constructs:
- Solely the plasmid containing the indigo pathway
- the plasmid containing the indigo pathway and an empty plasmid coding only for the same antibiotic resistance as the wiffleball plasmid with the same induction (IPTG), but not the wiffleball genes themselves
- the plasmid containing the indigo pathway and the plasmid containing the genes for the wiffleball.
- Indigo plasmid induced with 0.1 mM IPTG (spectinomycin as antibiotic in the medium)
- Indigo plasmid induced with 0.04 mM IPTG (spectinomycin as an antibiotic in the medium)
- Indigo and pTrc99A plasmid induced with 0.04 mM IPTG (spectinomycin and ampicillin as antibiotics in the medium)
- Indigo and wiffleball plasmid induced with 0.04 mM IPTG (spectinomycin and ampicillin as antibiotics in the medium)
- Indigo and wiffleball plasmid induced with 0.04 mM IPTG, but only one antibiotic in the medium (spectinomycin)
- Indigo, wiffleball and ncAA synthetase together were also tested in this experiment. However, it is not relevant to this specific engineering success cycle.
Once induced, samples were put into an incubator at 18°C while shaking at 200rpm. The assay was performed at 18°C as previous assays have shown that wiffleball formation has worked best for us at this temperature. We know that the first molecules of indirubin are produced after 24 hours, and because of that, we started to take samples of each flask after mentioned 24 hours. After that, we took new samples every six hours for the next 48 hours. Samples were collected by filling 1,5ml of each flask into separate collection tubes, which were then spun down for two minutes at 18500rpm and 4°C. The supernatant was discarded, and samples were frozen at -20°C until the assay was finished.
To evaluate our samples, pellets were resuspended in 300 µl KPI buffer and then sonicated to disrupt the cells. Afterwards, samples are spun down for 20 minutes at 18500 rpm and 4 °C. The supernatant is, again, discarded, and the pellet is resuspended with 1ml of DMSO. 200 µl of each sample was loaded on a 96-well plate and analysed in a plate reader by checking our sample's absorption at 540nm wavelength. Results were compared to a calibration curve of indirubin performed earlier to determine the indirubin concentration of each sample.
Learn
When evaluating the essay, we found that bacteria containing fully functional wiffleball plasmids and the indigo pathway have produced higher yields of indirubin compared to bacteria that contained the plasmid with the same resistance and induction as the wiffleball plasmid. However, they still produced less than those bacteria that only contained the indigo plasmid. In conclusion, the yields produced by bacteria containing the indigo and BMC plasmids were lower than expected (see figure 1).
Figure 1: Comparison between BL21 pCDFDuet-1 sTAF-sXTB with and without wiffleballs, pTrc99A plasmid-control and ncAAs. The pathway was shifted towards Indirubin production through L-Cysteine addition. A: Schematics of the used plasmids; B: Indigo concentration measured via fluorescence (extinction 612 nm, emission 670 nm); C: Indirubin concentration measured via absorbance at 540 nm. Production with 200 mL cultures in 500 mL Erlenmeyer-flasks, at 18 °C after IPTG-induction at OD = 0.8, with L-Tryptophan concentration of 2.5 mM, L-Cysteine concentration of 3 mM, ncAA-amount of 41.24 mg pAzF and with IPTG concentration shown in legend. Photometric readout using a plate reader.
Bacteria that contained two plasmids were treated with two antibiotics, as the second plasmid codes for a different resistance. As a result, it seemed as if the bacteria with two plasmids experienced more metabolic stress and reduced the production of our target molecule. This might explain why the bacteria containing two plasmids performed worse than the ones containing only one plasmid, which in turn were only treated with one antibiotic. Our mentioned assay has confirmed this assumption, as we can see, that samples only treated with spectinomycin have produced notably higher yields than the equivalent construct/condition treated with both antibiotics. Unfortunately, we were not able to push the production above the yield of our reference construct (sTAF-sXTB 0,1mM IPTG). In conclusion, the yield improvement by the wiffleballs was still lower than expected. We inferred that this may be due to the fact that the concentrations of pathway enzymes and wiffleballs do not fit together to get the maximal number of enzymes catched while not overproducing wiffleballs. But because of both Indigo pathway and wiffleball being induced by IPTG, it was not possible for us to regulate the production of Indigo enzymes and wiffleball proteins separately from each other, limiting our experimental freedom immensely and thus, making it impossible to optimise the pathway further this way.
Re-design
To solve this issue, we have exchanged the lac-promotor of the wiffleball plasmid with the tet-promotor which is inducible through doxycycline. This allows us to control the expression of both plasmids separately, thereby giving us the possibility to finetune the relations between wiffleball forming proteins and Indigo producing enzymes.
image of plasmid maps with lac promotor and with tet promotor
Re-build
To exchange the lac-promoter for the tet-promoter we used Gibson Assembly.
Figure 2: Comparison between pathway + wiffleball constructs (BMC), either with both IPTG-inducible promoter or both different promoters. Wiffleballs have either pLac promoter (IPTG-inducible) or tet-promoter (doxy-inducible), different doxycyclin concentrations are tested with consistent IPTG concentration of 0.04 mM. The pathway was shifted towards Indirubin production through L-Cysteine addition. A: Schematics of the used plasmids; B: Indigo-concentration measured via fluorescence (extinction 612 nm, emission 670 nm); C: Indirubin-concentration measured via absorbance at 540 nm. Production with 200 mL cultures in 500 mL Erlenmeyer-flasks, at 18 °C after IPTG-induction at OD = 0.8, with L-Tryptophan concentration of 2.5 mM and L-Cysteine concentration of 3 mM. Photometric readout using a plate reader.
Test again
After successfully changing the promotor via Gibson assembly, we have done an assay where we checked different doxycycline concentrations while analysing their effect on the yield of our target molecules. The following conditions were tested:
- BL21 sTAF-sXTB 0,1mM IPTG
- Bl21 sTAF-sXTB + wiffleball_tp (tet-promotor) 0,04mM IPTG, 10ng/ml Doxycycline
- Bl21 sTAF-sXTB + wiffleball_tp 0,04mM IPTG, 20ng/ml Doxycycline
- Bl21 sTAF-sXTB + wiffleball_tp 0,04mM IPTG, 50 ng/ml Doxycycline
- Bl21 sTAF-sXTB + wiffleball_tp 0,04mM IPTG, 75 ng/ml Doxycycline
- Bl21 sTAF-sXTB + wiffleball (old promotor) 0,04mM IPTG
Learn
Already during the assay, we have seen that the samples containing the new tet promotor did not produce any Indirubin. Additionally, the flasks did not appear to grow denser after induction and the pellets of our centrifuged samples showed a very light, almost white colour. Additionally, our assumption that these samples were unable to produce our target molecule was confirmed by the evaluation of this assay (see figure 2). Until now, we were not able to definitely determine why the change of the promotor ended in the mentioned results. Our running assumption so far is that something might have gone wrong during cloning, impeding the function of the Tetracycline resistance required for constructs that are induced through Doxycycline, resulting our bacteria kicking the bucket. However, the sequencing results for the tet promotor appeared to be correct, speaking against this assumption. We are going to look further into this, trying to understand from where this problem emerges.
Re-design again
For the time of this writing, we were not able to make any changes to the promotor so far. However, our planning to proceed is as follows:
In case we find the reason for the inhibiting effect of the promotor, we would want to change the problematic sequence. However, in case we might not be able to find a reason, we could also exchange the entire promotor with the araBAD promotor, which is induced by Arabinose. The procedure to achieve this change would likely be by Gibson assembly.
With the then again changed wiffleball promoter, we would repeat the production assay again, testing different inducer concentrations to find out, if the yield increases and our initial thought was right, that the concentrations of pathway enzymes and wiffleball enzymes hindered better yield.
BMCs engineering success
Cycle 1
Design
An important part of our project was engineering and optimizing microcompartments for bacteria. This means that the microcompartments had to be expressed in the bacteria, catch enzymes to their inside and assemble into the right conformation. The Kerfeld group, who inspired part of our work through their publication [3], kindly shared their wiffleball and fluorescent protein constructs with us. Initial screenings and assessments of the formed compartments seemed easiest by using fluorescence microscopy. The fluorescent proteins are both fused to a catcher (mVenus2 to the SpyCatcher, mTurquoise2 to the SnpCatcher) that specifically recognizes a tag on the internal loop of the T1 protein of the wiffleball (SpyTag and SnpTag, respectively) and binds to it, leading to the formation of a peptide bond. With this protein ligation system, the proteins can be localized inside the microcompartments.
Our initial experiments had three goals:
- show that the catching works (Fig. 3). For this we used both, untagged and tagged versions of the wiffleball T1 protein, and co-expressed them with mVenus2. Furthermore, we decided to complement our microscopy data with Western blot data (Fig. 4 + 5). For this, we took advantage of the presence of a His-tag on the T1 protein and used an anti-His antibody to detect it
- test different induction and incubation conditions to find the optimal settings to express properly formed wiffleballs
Build
We prepared the samples for our initial microscopy screening by double-transforming BL21(DE3) cells with the wiffleball plasmids with or without the tags and the plasmid expressing mVenus2 also tagged.
Test
Liquid cultures were induced with varying concentrations of doxycycline (50 and 200ng/ml) and IPTG (50 and 100µM) and cultivated for 24h at 18°C. After incubation, cells were visualized using fluorescent microscopy and harvested for western blots.
Figure 3: Fluorescent microscopy of T1 catching the mVenus2, when the minimal or full wiffleball construct is expressed; A: Controls for the induction; B: T1 with and without the Spy/Snp tags; scale bar, 5µm.
Figure 4: Figure 1.4 in results: Western Blot showing the formation of the peptide bond between the T1 protein and mVenus2 in cells expressing the minimal wiffleball with and w/o tags. The detection of the T1 protein was performed with an anti-his antibody.
Figure 5: Figure 1.5 in results: Western Blot showing the formation of the peptide bond between the T1 protein and mVenus2 in cells expressing the full wiffleball with and w/o tags. The detection of the T1 protein was performed with an anti-his antibody.
Learn
Through the results of this initial round of screening, we found that the mVenus2 localizes to fluorescent foci in the presence of the tagged version of the T1 protein. This localization needs the SpyTag on mVenus2. We could confirm this observation by detecting a higher molecular weight band for the his-T1 protein in the Western blot. We also observed that the minimal and full wiffleballs behave differently. In case of the full wiffleball, more fluorescent foci are found in the cells than in the case of the minimal wiffleball. Also, we observed that the combination of inducers that worked best was 100µM IPTG together with 50ng/ml doxycycline.
After evaluating our first round of experiments, we were eager to find out how to further optimize our compartmentalization system.
Cycle 2
Design
We opted for trying out higher IPTG concentrations to see if we could quantitatively optimize the production of the compartments.
To further enhance the system, we decided to test the wiffleballs in different strains, since the inherently different genetic backgrounds of different E. coli strains can lead to significant differences in expression and assembly of the compartments.
Build
We transformed our plasmids into competent MG1655 cells in the same combinations as explained in cycle 1.
Test
Liquid cultures were induced with varying quantities of IPTG (100, 400, 700, 1000µM). After 24h incubation at 18°C, cells were visualized using fluorescence microscopy (Fig. 6) and harvested for Western blots (Fig. 7). For further quantification of our data, we decided to manually count the fluorescent foci formed within the bacteria under different IPTG concentrations. While we wanted to assess the formation of the fluorescent foci(Fig. 8), we also needed to investigate the viability of the cells, since it is important for our system not to negatively impact bacterial growth. Hence, we generated growth curves for all our samples and compared them (Fig. 9). For higher reliability, we repeated these experiments three times for BL21(DE3) and twice for MG1655.
Figure 6: Fluorescent microscopy of different induction concentrations for the BMC expression with IPTG in MG1655 mVenus2 was always induced with 50ng/ml Doxycycline; scale bar, 5µm
Figure 7: Western Blots of different induction concentrations for the BMC expression with IPTG in MG1655. The 100µM IPTG 0ng/ml DOX sample of the pellet and supernatant had been switched, when loading the SDS-gel.
Figure 8: Quantification of the foci of the full wiffleball visible under the microscope with different induction concentrations of IPTG for the BMC expression under the microscope in MG1655; n=2.
Figure 9: MG1655 growth in plate reader at 30°C with different induction conditions. In legend: Wiffleball (IPTG (µM)/doxycylcine (ng/ml)).
Learn
The Western blot revealed that MG1655 cells barely expressed the full wiffleball. There were only few fluorescent foci in the cells. Additionally, the cells seemed to be impacted in their growth at higher IPTG concentrations. The expression of the T1 protein in the context of the minimal wiffleball construct was overall higher and detectable by Western blot. We note here that in the Western blot we can only detect (1) expression of the T1 protein and (2) whether the protein of interest (e.g. mVenus2) has been ligated to it. This assay is not the appropriate one to assess the formation of the compartments. Higher IPTG concentrations didn’t seem to influence bacterial growth. The minimal wiffleball always needed higher inducer concentration compared with the full wiffleball.
However, the change of strain did not show any significant advantage. We therefore decided to try out other strains.
Cycle 3
Design
The strain ME5119 is a genome-reduced derivative of MG1655 [4][5] We hoped that, by being genome-reduced, less energy would be needed for the expression of housekeeping proteins, and therefore this strain would be more effective at synthesizing and assembling the wiffleballs. While looking for genome-reduced E. coli strains, we found another genome-reduced strain derivative of MG1655, MDS69, in particular the LowMut7 commercial version from Scarab Genomics. We decided to repeat the cycle above with the new strains.
Build
We made competent cells of the ME5119 strain, bought competent MDS69 LowMutT7 cells from Scarab Genomics and transformed them both with the wiffleball constructs and the mVenus2 plasmid.
Test
Figure 10: ME5119 growth in plate reader at 30°C with different inducer conditions. In legend: Wiffleball (IPTG (µM)/Doxycylcine (ng/ml))
Figure 11: LowMut T7 growth in plate reader at 30°C with different inducer conditions. In legend: Wiffleball (IPTG (µM)/Doxycylcine (ng/ml))
We prepared overnight cultures of the different strains and induced them with IPTG and doxycycline. Again, fluorescence microscopy was conducted, followed by a confirmatory Western blot. We also performed a growth curve assay with both strains (Fig. 10 + 11) . While we repeated all these experiments twice with ME5119, we could perform the microscopy only with MDS69 LowMutT7, due to time limitations (Western blot results will come after the wiki-freeze).
Learn
Unfortunately, none of the genome-reduced strains showed a better result in the microscopy experiments than the BL21 strain. The fluorescent foci detected in the previous cycles could not be observed and no bands could be detected in the Western blots. However, the growth curve of the MDS69 LowMutT7 strain looked much more promising (Fig. 10 + 11), and we hypothesized that inducing and letting the cells grow at low temperatures would hinder the expression of the wiffleballs. We therefore decided to perform another round of experiments with MDS69 LowMutT7. By talking to Cheryl Kerfeld, an expert on BMCs and on the wiffleball in particular, whose work greatly inspired our project, we received valuable advice on changing our induction temperatures. We changed the temperature from the constant 18°C that we previously used to an initial 3 hours at 37 °C with subsequent lowering to 18 °C for another 16 hours. Since changing the temperatures of induction and growth seemed to have great impact on the expression in experiments performed by the Kerfeld lab, we decided to also include BL21(DE3) again in our next round of experiments, given that they had initially given better results than all other strains.
Cycle 4
Design
We decided to investigate how this change of temperature would affect the formation of the wiffleballs, and therefore induced MDS69 LowMutT7 and BL21(DE3) with the same conditions as always. We also wanted to find out what happens when we incubate the bacteria for even longer, 48 hours in total, whether it would affect the wiffleballs (degradation, aggregation, changes in expression levels).
Build
We used our bacteria from cycle 1 and 3.
Test
We prepared overnight cultures of the different samples and induced them with IPTG and doxycycline. As described above, this time we started with a 3-hour incubation at 37°C and continued with 16 hours of incubation at 18°C. After 24 hours, fluorescence microscopy was conducted –and will be followed by a confirming Western blot after the wiki freeze. Afterwards, we returned the samples to the incubator and repeated the experiments after 48 hours.
Learn
We found these conditions to dramatically improve wiffleball expression after 24 hours, registering a significant increase in the number of fluorescent foci in our bacteria in both strains. In our samples that were incubated for 48 hours, we observed even more fluorescent foci than in our previous experiments, especially in the bacteria containing the minimal wiffleball construct. In this case, the localization was not limited to the poles of the bacteria, but was more distributed throughout the cells.
We will confirm our results by Western blot before the grand jamboree.