Preliminary experiments

Nanoparticle synthesis by sequential addition of AgNO₃ and HAuCl₄ using WT Escherichia coli BL21

1. Testing nanoparticle production by a liquid culture of WT E. coli BL21 in a 96-well plate

Dates: 07.07-09.07

Duration: 2.5 days

Goal: Check (through absorbance) whether we can obtain bimetallic nanoparticles by adding silver and gold ions at the same time. We also aimed to check if adding different concentrations would influence the absorbance readings.

Method: Test the influence of [HAuCl₄]:[AgNO₃] ratio (1:10, 1:15, 1:20, 10:1, 15:1, 20:1) and various HAuCl₄ concentrations on nanoparticle production with a cell system (liquid culture of E. coli BL21) by measuring absorption spectra. For this experiments, the salts were added simultaneously

Hypothesis: According to literature, 10:1 ratio is the most optimal¹
while the concentration for a biological system is 1 mM of AgNO₃ ².

Protocol: Nanoparticle Synthesis by Sequential Addition of AgNO3 and HAuCl4 Using Escherichia coli BL21 Wild Type

Results: Raw Absorbance Data, TEM pictures

Conclusion: Most absorbance measurements exceeded the reliability threshold of 1-1.2, so the measurements were discarded. Our team was not aware of this threshold until intervention by supervisors, which occurred only after the first week of experiments. The TEM pictures showed that the used concentrations were too high, so higher dilution was necessary.

Discussion:

• The samples prepared in the well plates were quite concentrated and often the samples were difficult to visualize under the microscope. So, use flasks to have diluted samples.
• 1 mM of Gold provided some good results. However, the nanoparticles were quite heterogeneous in size. So, we use this concentration in flasks because we obtained good micrographs. Additionally, we use 10 mM gold to check for differences between a low and a high concentration of gold. We also did this experiment for silver.
• Addition of 1Ag:15Au resulted in rather large clusters of nanoparticles and often an accumulation of salts with some odd morphologies. Similar results were obtained for 1Au:20Ag. Gerda suggested we first add silver and once we obtain the nanoparticles we can add gold ions to check if the morphologies are influenced.

2. Testing nanoparticle production by a liquid culture of WT E. coli BL21 in a flask

Dates: 12.07-18.07

Duration: 6 days

Goals:

• Test synthesis in flasks instead of a 96-well plate.
• Test lower (1 mM) and higher (10 mM) concentrations for both HAuCl₄ and AgNO₃.
• Test sequential addition of gold after silver to see if the morphology of the nanoparticles changes.

Method: Carry out nanoparticle synthesis with the different conditions in 50 ml flasks over a span of 3 days. Measure the absorbance twice a day.

Hypothesis:

• Lower concentrations of the salts should yield less agglomerates on the TEM pictures.
• Shaking in the flask should prevent aggregation.
• The morphology is expected to change upon adding the gold to the synthesized silver nanoparticles.

Protocol:

• Preliminary Flask Experiment
• Continuation of Preliminary Flask Experiment and Setting up Secondary Experiments

Notes:
*We took out 500 ul for measurements after 3 days, so the concentrations are not a 100% correct

Results

Raw Absorbance Data

Fig. 1 | Absorbance measurements of wildtype E.coli BL21 in combination with 1mM HAuCl₄ and 1 mM AgNO_3. After 1, 2, and 3 days with two measurements per day.

Fig. 2 | Absorbance measurements of wildtype E.coli BL21 in combination with 10mM HAuCl₄ and 10 mM AgNO_3. After 1, 2, and 3 days with two measurements per day.

Conclusion:

• Only absorbance measurements were obtained for this experiment, so no conclusive results were obtained about the aggregation of nanoparticles in the samples.
• Results show that after 3 days suddenly a peak forms at ~650 nm. We do not know what this is attributed to, but it could be caused by the fact that gold has a higher oxidation capacity. That means that, upon addition of HAuCl₄, the reducing agents will first have to reduce all the gold ions. After this, silver salts will again start being reduced. As shown in Cheng et al., 2012, golden nanoparticles can serve as nucleation points for silver to form spikes¹. Perhaps this is the mechanism by which such a shift can occur after 3 days.

3. Testing different sonication settings for obtaining a cell-free system for silver nanoparticles synthesis

Date: 20.07-23.07

Duration: 3.5 days

Motivation: Our goal was to obtain a cell-free system for nanoparticle production, so we wanted to test sonication as a possible technique to obtain a cell-free system for bioreduction.

Goal: Test nanoparticle production with a system of WT E. coli BL21 lysed by sonication.

Method: Perform nanoparticle synthesis with such a sonicated biological system. Test different sonication settings to find an optimum (20/30/40s x2). Compare a cell system to supernatant to cell debris and LB medium.

Hypothesis: Longer sonication would lead to lysis of more cells and, therefore, to a higher total concentration of reducing agents in the sample. This would lead to a higher nanoparticle yield and, therefore, higher absorbance values.

Protocol: Testing Different Sonication Settings for Obtaining a Cell-Free System for Silver Nanoparticle Synthesis

Notes:

• It is ideal to test whether the system is indeed cell-free and this is done by plating out the culture after sonication. However, upon plating these cultures, we found that these cultures result in bacterial growth. The sonication process is not sterile and thus it's very likely that the cell-free extract is contaminated. (Fig. 3)

Fig. 3 | Plates showing bacterial growth after sonication

Results:

Raw Absorbance Data

Fig. 4 | Absorbance measurements after 0h of cell free extract after different sonication settings. Sonication settings were 20s, 30s, or 40s. Different concentrations of silver were tested in combination with different volumes of cell free extract.

Fig. 5 | Absorbance measurements after 72h of cell free extract after different sonication settings. Sonication setting were 20s, 30s, or 40s. Different concentrations of silver were tested in combination with different volumes of cell free extract.

Fig. 6 | Absorbance measurements of components of cell system after exposure to different concentrations of silver after 0h. The different concentrations of silver were: 1 mM, 5 mM, and 10 mM.

Fig. 7 | Absorbance measurements of components of a cell system after exposing to different concentrations of silver after 72h. The different concentrations of silver were: 1 mM, 5 mM, and 10 mM.

Conclusion:

• Sonicating the liquid culture is not sufficient to create a sterile, cell-free system. It is therefore necessary to filter-sterilize the supernatants prior to bioreduction.

4. Testing the capacity of different surfactants to prevent aggregation of nanoparticles during synthesis with a supernatant of WT E. coli BL21 grown on LB-Nitrate.

Dates: 09.07-12.07

Duration: 3.5 days

Goal: Prevent aggregation of nanoparticles during synthesis by using surfactants.

Method: Add surfactant prior to beginning nanoparticle synthesis. The tested surfactants were: Triton X-100, Tween 20, SDS, Nonidet.

Hypothesis: No review was done on whether one surfactant should perform better than others. The point of the experiment was to see whether this step would have any beneficial effect on preventing agglomeration of nanoparticles.

Protocol: Testing Four Surfactants and Concentrations

Notes:

• We added the 0.5 mM of silver to limit the risk of obtaining absorbances above 1 resulting in the need for dilutions

Results:

Raw Absorbance Data

Fig. 8. | Absorbance measurements of BL21 supernatant grown on LB-nitrate with addition of different surfactants. The added surfactants were: nonidet, SDS, trition, and tween. The surfactants were added prior to starting the nanoparticle synthesis. After which, 1mM silver salts were added.

Fig. 9. | Absorbance measurements of BL21 supernatant grown on LB-nitrate with addition of different surfactants. The added surfactants were: nonidet, SDS, trition, and tween. The surfactants were added prior to starting the nanoparticle synthesis. After which, 1mM silver salts were added. Absorbance measurements were done after 72h.

Fig. 10. | Absorbance measurements of BL21 supernatant grown on LB-nitrate with addition of different surfactants. The added surfactants were: nonidet, SDS, trition, and tween. The surfactants were added prior to starting the nanoparticle synthesis. After which, 1mM silver salts were added. After 72h, also 0.5 mM gold salts were added.

Fig. 11. | Absorbance measurements of BL21 supernatant grown on LB-nitrate with addition of different surfactants. The added surfactants were: nonidet, SDS, trition, and tween. The surfactants were added prior to starting the nanoparticle synthesis. After which, 1mM silver salts were added. After 72h, also 0.5 mM gold salts were added. Absorbance measurements were performed 48h after adding the gold salts.

Conclusion: The obtained absorption spectra did not point at any benefit of using surfactant. Due to the restricted lab time, it was decided to proceed without using surfactants in the following experiments.

Nanoparticle synthesis by simultaneous addition of AgNO₃ and HAuCl₄ using WT E. coli BL21

5. Test nanoparticle synthesis with supernatants of liquid cultures grown in three different broths.

Date: 12.08-15.08

Duration: 3.5 days

Goal: Test the influence of growing WT E. coli BL21 in different media on the bioreducing qualities of the supernatant

Method: Use supernatants of liquid cultures of WT E. coli BL21 grown in 3 different media (LB, MH broth, TSBS) in combination with salts of gold (HAuCl₄) and silver (AgNO₃) to produce nanoparticles.

Hypothesis: Based on literature review, MH Broth is the most promising medium².

Protocol: Three Broths Cell Free

Notes:

• Redo MH sample absorbance measurements with dilution 6x, because some measurements were too high

Results:

Fig. 12 | Absorbance measurements of wild type E.coli BL21 supernatant grown on different media in combination with silver (0.3 mM) and gold (3 mM) salts. The different media were: 2xYT, LB, MH broth, and TSBS.

Fig. 13 | Absorbance measurements of different media in combination with silver (0.3 mM) and gold (3 mM) salts. The different media were: 2xYT, LB, MH broth, and TSBS.

Raw Absorbance Data

Discussion:

• Growth media, once again, show a higher absorbance and better peaks. To test what this could be attributed to, we eventually set up Supplementary Experiments 8 and 9.
• MH does show the highest absorbance, which corroborates the hypothesis
• The experiment did not yield good results after 3 days in the shaker. In order to save time and space, we proceeded to conduct experiments for 24h and 24-well plates.
• This experiment will be repeated in a 24-well plate (see Core Experiment 1)

6. Compare nanoparticle synthesis with the supernatant of WT E. coli BL21 grown either in LB-Nitrate or in LB.

Date: 09.08-11.08

Duration: 2.5 days

Goal: Identify if the addition of nitrate to the growth medium has a beneficial effect on the synthesis of nanoparticles.

Method: Test if the supernatant obtained from WT E. coli BL21 grown on LB-Nitrate performs better than the supernatant obtained from LB in the synthesis of nanoparticles

Hypothesis: As seen in the reference paper, LB-Nitrate supernatant should contain more reducing agents (primarily nitrate reductases), which will accelerate the formation of nanoparticles.

Protocol: LB vs LB-Nitrate

Notes:

• 10.08
  • L3 sample - the cotton cap was off, so possibly contaminated
  • Noticed (because it was wrong in the table of 09.08) that L3 & N3 had BL21 instead of LB/LB-Nitrate, so remake those to be measured after 21h (Last time was done 17:00-14:00)
• 11.08
  • L1 - The cotton cap was off
  • L3 - the cotton cap was off again

Results:

Fig. 14 | Absorbance measurements of wild type E.coli supernatant grown on LB or LB-nitrate in combination with silver (0.1mM) and gold (1mM) salts after 24 hour.

Fig. 15 | Absorbance measurements of wild type E.coli supernatant grown on LB or LB-nitrate in combination with silver (0.1mM) and gold (1mM) salts after 48 hour. Samples were diluted 10 times and medium was removed since it was contaminated.

All TEM pictures

Raw Absorbance Data

Discussion:

• The results after 24h were diluted too much, so the absorbance measurements are outside of the range of reliable values. These results could therefore not be used. Moreover, the LB control was contaminated.
• Both LB-Nitrate and supernatant of E. coli BL21 grown on LB-Nitrate showed a higher absorbance at the peak, which points to the fact that nitrate-containing medium has a beneficial effect on nanoparticle yield/rate of formation.

Chemical synthesis

7. Testing different [HAuCl₄]:[AgNO₃] ratios for ascorbic acid synthesis

Date: 29.07

Duration: 0.5h

Goal: Identify the relevant range of [HAuCl₄]:[AgNO₃] for ascorbic acid modeling experiments.

Method: Due to the fact that we wanted to do modeling with a biologically relevant pH range 5-9. And the paper¹ that we used as a reference used pH 3, we had to identify a range of ratios that would be tested within the Modeling experiment.

Hypothesis: We expected the ratio to not be equal to 10:1, since the reducing capacity of ascorbic acid is dependent on the pH.

Protocol: Chemical Synthesis

Results:

Fig. 16 | Absorbance measurements of nanoparticles formed using ascorbic acid with six different gold and silver concentrations (n = 3). The highest average absorbance at 800 nm was found for a ratio of 0.20 mM gold and 0.10 mM silver.

Conclusion: It was not clear what the optimal ratio was.

8. Luminol-mediated addition of spikes to spherical silver nanoparticles

Date: 25.07-01.08

Duration: 6.5 days

Goal: Synthesize luminescent dendritic gold and silver nanoparticles using luminol.

Method: First use a liquid culture of WT E. coli BL21 to produce spherical silver nanoparticles during 3 days, then add luminol and HAuCl₄ to induce the formation of golden spikes on these spherical nanoparticles.

Hypothesis: We expected to obtain dendritic gold and silver nanoparticles.

Protocol:

• Luminol Experiments
• Luminescence experiment

Notes:

Luminol experiment:

• The absorption spectrum was measured in the 325-1100 nm range. LB medium was used as blank to dilute the samples. Dilution was done to obtain absorbance values of a maximum of 1.00
• Dilution factors were:
  • Experiments (flasks) 1-4: 10x
  • Experiments 5-7: 20x
• Experiment 8: 25x

Luminescence experiment:

• 02.08: In LE1 & LE5: 0,88 mg of luminol in each sample--> 12 mg luminol in luminescence protocol → 0,88/12=0,073 = factor with which other quantities are calculated.
• 03.08:
  • 1/10 out of each sample-->Dilute 10x
  • For 10x buffer dilution
  • 9 ml buffer + 1 ml luminol experiment needed per sample
  • 9*9= 81 ml buffer needed
  • About 100/20=5 ml buffer + 95 ml water

Results:

Raw Absorbance Data

Fig. 17 | Absorbance measurements of wildtype E.coli BL21 in combination with luminol and silver and gold salts.

No quantifiable results are provided for the Luminescence experiment, however no luminescence was observed

Conclusion: Contrary to our expectations, this experiment did not yield luminescent dendritic nanoparticles. Therefore, the luminol research line was not continued. However, theoretically it is a very interesting field for research.

References:

1. Cheng, L. C. et al. Seedless, silver-induced synthesis of star-shaped gold/silver bimetallic nanoparticles as high efficiency photothermal therapy reagent. J Mater Chem 22, 2244–2253 (2012).
2. Gurunathan, S. et al. Biosynthesis, purification and characterization of silver nanoparticles using Escherichia coli. Colloids Surf B Biointerfaces 74, 328–335 (2009).

Core experiments:

1. Establishing a methodology for obtaining WT E. coli BL21 supernatant for nanoparticle production

Date: 15.08-17.08 and 23.08-25.08 (repeated in triplo)

Duration: 1.5 days

Goals:

• Identify the growth medium that yields a supernatant of WT E. coli BL21 that is most suitable for producing nanoparticles.
• Determine whether the cell lysate should be included in the supernatant to achieve optimal nanoparticle production.

Method: Use supernatants of liquid cultures of WT E. coli BL21 grown in 4 different media (LB, LB Low Salt, MH Broth, TSBS) in combination with salts of gold (HAuCl₄) and silver (AgNO₃) to produce nanoparticles. Also test whether lysing the liquid culture prior to obtaining the supernatant is beneficial.

Hypothesis:

• Based on literature review, MH Broth is the most promising medium¹.
• Lysed supernatant will have a better capacity at producing nanoparticles, as it also contains reducing agents that are present inside the cell.

Protocols:

• Compare Supernatants of 5 Different Broths
• Compare Supernatants and Lysates of 4 Different Broths

Results:

Fig. 18 | Absorbance measurements of wild type E.coli BL21 supernatant grown in different media in combination with silver and gold salts. The different media tested were: LB, LB low salt, MH broth, and TSBS. Measurement done after 24 hour.

Fig. 19 | TEM pictures of nanoparticles formed in two different media using supernatant. A) Three TEM pictures formed in LB medium. Aggregates are still formed, but distinctive nanoparticles can be identified. B) Three TEM pictures of nanoparticles formed in MH medium. No aggregates are observed, formation of urchin-like nanoparticles.

Raw Absorbance Data

TEM Pictures

Conclusion:

Based on the absorbance measurements at 800 nm (Fig. 18) and the TEM pictures, the highest absorbance, paired with less agglomeration, was observed for non-lysed MH supernatant. Therefore, for the following experiments using supernatant for nanoparticle production, proceed with MH medium and do not lyse the cell culture prior to obtaining the supernatant.

Discussion:

It was noticed that higher absorbance is not equal to a better quality of the sample. This could be caused by the accidental absorbance of nanoparticle agglomerates. Formation of such agglomerates is undesired for the application in PTT, since agglomerates of bigger sizes could have a much higher toxicity. It is, therefore, always advised to use absorbance measurements in combination with TEM micrographs to evaluate the quality of the produced nanoparticles.

The spectra also showed the ability of media to convert salts of metals into nanoparticles. This was an unexpected discovery, so Supplementary Experiments 8 and 9 were set up to determine which constituents of the media were responsible for producing the nanoparticles.

Potential explanations for the fact that the cell lysate performs worse than the non-lysed supernatant are:

• 1. Lysis disturbs the reducing agents and decreases their capacity to convert salts into nanoparticles
• 2. Cell lysate contains molecules that interfere with the reducing action of present reducing agents. This can be caused by these molecules having a lower oxidizing potential than the salts or by their detrimental effect on the functionality of the reducing agents as such.

2. Modeling #1: Determining the optimal HAuCl₄:AgNO₃ ratio for PTT-optimized nanoparticle production with ascorbic acid

Date: 10.08

Duration: 4h

Goal: identify which ratio of HAuCl4:AgNO3 is optimal for the production of nanoparticles with the highest absorbance at 800 nm, using ascorbic acid.

Method: use chemical synthesis with ascorbic acid to test the following ratios: , and compare them based on the intensity of absorbance at 800 nm.

Hypothesis: based on literature, the ratio of 10:1 is optimal².

Protocol: Chemical Synthesis of Nanoparticles Using Ascorbic Acid Modeling

Notes: We noticed that if the time in between adding AgNO3, HAuCl4, and Ascorbic Acid varied a lot, that the color of the solution also varied (in between replicates). So we tried to make sure there was very little variation.

Results:

Fig. 20 | Absorbance measurements of chemical nanoparticles produced using ascorbic acid with different concentrations of silver and gold salts. Nanoparticles were produced at different pH: A.) pH 5, B.) pH 7, C.) pH 9.

These absorbance plots were used to obtain an interactive model, which can be found on the Engineering page and the Modeling page.

Formatted Absorbance Data

TEM Pictures

Conclusion: 1.85:1 is the optimal molar ratio of HAuCl4:AgNO3 for the synthesis of nanoparticles with the highest absorbance at 800 nm. Therefore, in all following experiments this ratio is used. Further discussion of the methodology and the implications of the results are provided on the Modeling page.

3. Determining the relevant range of HAuCl₄ concentration for modeling experiments in a biological system

Date: 16.08-19.08

Duration: 2.5 days

Goal: identify which concentrations of HAuCl₄ should be used for the modeling experiments with supernatants of WT E. coli BL21 grown on MH.

Method: Obtain absorbance measurements for biologically synthesized nanoparticles in range of concentrations of HAuCl₄ = [0,2,4,6,8] mM and establish the adequate range for modeling.

Hypothesis: From preliminary experiments we saw that a concentration of 10 mM for HAuCl₄ did not have a beneficial effect on nanoparticle formation. Therefore, the optimum was expected to lay between 1 and 8 mM.

Protocol: Determining the Relevant Range of HAuCl4 Concentration for Modeling Experiments in a Biological System

Notes:

• 18.08: experiment was conducted, but the wells were diluted with different amounts, which rendered the data useless. Repeat the set up today + in triplo

Results:

Fig. 21 | Effect of HAuCl₄ concentration on the absorption spectrum of biologically synthesized nanoparticles. Experiments were performed with Escherichia coli BL21 supernatant and varying concentrations of HAuCl₄. The silver:gold salts ratio was 1:1.85.

A, Full absorption spectrum, dotted lines represent standard error (n = 3). B, Barplot of the A_{800 nm} for the same measurements. Error bars represent standard error (n = 3).

Raw Absorbance Data

Conclusion: The highest absorbance was obtained in the range below 8 mM. Therefore, for the subsequent modeling the adequate range was established to be 2-6 mM HAuCl₄.

4. Modeling #2: determining the optimal synthesis conditions for nanoparticle production using a biological system

Date: 24.08-26.08

Duration: 1.5 days

Goal: identify the optimal synthesis conditions (pH, T°C, [HAuCl₄]) for producing nanoparticles with a high absorbance at 800 nm, using a supernatant of WT E. coli BL21 grown in MH. Various optimization objectives were analyzed.

Method: use 3 equidistant values for the three variable conditions to identify the optimum combination with the help of Box-Behnken design.

Hypothesis:

• Lower pH is more optimal, since the concentration of hydroxide ions, which compete with other oxidation agents, is lower
• Based on literature, higher temperature is more optimal³ .
• Based on literature, gold concentration should have local optimum²

Protocol: Differing pH, T(°C), [Au3+] modeling

Notes:

• It was quite difficult to use the pH-calibrating machine when adjusting the pH of the media

Results are provided on the Modeling page, due to the verboseness of the obtained results for all optimization objectives.

Formatted Absorbance Data

Conclusion: Synthesis conditions have been optimized for a variety of objectives, which might depend on the production process prerequisites. An enumeration of the results and implications of those are discussed on the Modeling page.

5. Testing the influence of copA, napA, cueO, melA E. coli genes on nanoparticle production

Date: 24.08-26.08

Duration: 1.5 days

Goal: test the influence of overexpressed ASKA genes (copA, napA, cueO, melA) on nanoparticle production.

Method: Compare the effect of adding a cell lysate that contains an overexpressed protein of interest on the properties of the produced nanoparticles.

Hypothesis: All these proteins have been shown to increase the reduction rate of silver nanoparticles, therefore a higher yield (shown as higher absorbance) is expected over the same time span.

Protocol: Testing the influence of copA, napA, cueO, melA E. coli genes on nanoparticle production

Notes:

• 18.08: Not enough supernatant was obtained from the uninduced cultures, so the 24 well plates had to be substituted for 96 well plates. Repeated in 24-well plate on 25.08

Results:

Fig. 22 | Bar plots showing the absorbance at 800 nm for samples of nanoparticles synthesized with supernatant of MH, with addition of overexpressed Escherichia coli proteins: CopA, CueO, MelA and NapA. A comparison was held between wildtype (control) and samples that contained the overexpressed proteins. Error bars represent standard error (n = 3). See Results pages for full spectra, including those of control samples.

Fig. 23 | Absorbance measurements of nanoparticles synthesized with supernatant of MH, with addition of overexpressed Escherichia coli proteins: CopA, CueO, MelA and NapA. A comparison was held between wildtype (control) and samples that contained the overexpressed proteins. Error bars represent standard error (n = 3).

Statistical Analysis

Raw Absorbance Data

Conclusion: The addition of lysate typically has a detrimental effect on the bioreducing capacity of the supernatant/medium. This could potentially be caused by undesired interactions with other oxidizing agents or by agglomeration of nanoparticles due to the presence of cell debris. The takeaway for future experiments is to translocate the expressed proteins into the extracellular space. This way, the lysis of the cells is not necessary.

6. Testing the effect of nitrate in the growth medium on nanoparticle production capacity of the liquid culture supernatant

Date: 24.08-26.08

Duration: 1.5 days

Goal: test if growth on MH-Nitrate has a beneficial effect on the supernatant used for nanoparticle production.

Method: Compare blends of all A/K/T pellet lysates in combination with supernatant of WT E. coli BL21 grown on MH with or without nitrate.

Hypothesis: based on literature, supernatants of a culture grown on a medium containing nitrate, will have a higher reducing capacity. Therefore, it is expected to yield better nanoparticles^1,4

Protocol: Compare A/K/T pellet lysate with supernatant of WT grown on MH-nitrate

Results: results can be found on the results page.

Raw Absorbance Data

Statistical Analysis

Conclusion: Nitrate in the medium does not bear a significant effect on nanoparticle synthesis when cell lysate is used

Discussion: Most probably, the lack of beneficial effect on nanoparticle production in Core Experiments 6 and 7 is caused by the addition of lysate into the bioreduction mix. This may perturb the reducing capacity of other molecules, which, in total, has a detrimental effect on nanoparticle production. In future, consider other options for overexpression of the protein in the extracellular space.

7. Testing IPTG-induced protein overexpression for ASKA

Date: 17.08

Duration: 6h + overnight culture

Goal: Show that ASKA and transformants contain plasmids, in which the production of a protein of interest is inducible with IPTG.

Method: Take samples from liquid cultures of the respective strains before and after induction with IPTG and prepare an SDS gel.

Hypothesis: Expect thicker bands than in uninduced at:

• CopA - 88 kDa
• NapA - 93 kDa
• MelA - 51 kDa
• CueO - 57 kDa

Protocol: Testing IPTG-induced protein overexpression for ASKA

Results:

Fig. 24 | SDS gel of four ASKA genes CopA, NapA, CueO, and melA. 1. ASKA-NapA non-induced. 2. ASKA-NapA induced with IPTG. 3. ASKA-CueO non-induced. 4. ASKA-CueO induced with IPTG. 5. Protein ladder. 6. ASKA-CopA non-induced. 7. ASKA-CopA induced with IPTG. 8. MalE non-induced. 9. ASKA-MalE induced with IPTG. 10. BL21 wild type. The ladder is added as a reference.

Conclusion: As seen in Fig. 24, all proteins showed a more significant band at the height of their respective molecular weight, meaning that induction with IPTG resulted in the overexpression of the protein.

8. Cloning experiments

Note: The following steps were repeated multiple times until it was seen in step 4 that the inserts were present and then everything was sent for sequencing.

1. Rapid ligation

Date: 27.07

Duration: 1 day

Goal: Clone gBlocks into pJET 1.2/blunt vector

Method: Use a rapid DNA ligation Kit and then transform into DH5α

Hypothesis: Due to the lethal restriction enzyme gene in the pJET vector, all surviving colonies should have the insert

Protocols:

• Rapid ligation
• Transformation

Results: All the plates had colonies, meaning that they all should have the correct insert.

1. Plasmid isolation

Date: 28-01.08

Duration: 3 days

Goal: Isolate the plasmids with the insert from the pJET 1.2 vector

Method: Using the plasmid isolation kit from Qiagen

Protocol: Inoculate cultures from plate

Follow the protocol in the plasmid isolation kit

Restriction digest

Hypothesis: seeing two bands on gel electrophoresis, indicating the insert and the backbone.

Results:

Fig. 25 | Gel electrophoresis for cup1, copA, and napA digested with NdeI and XbaI. 1-6. Digested cup1. 7. Digested copA. 8. Digested napA.

Conclusion: For cup1 only one band at around 3000 bp is seen. However the insert is only 100 bp long, so it cannot be seen on gel. For copA and napA, 2 bands can be observed. Indicating that the insert is present.

1. Gel isolation

Date: 01.08

Duration: 1h

Goal: Cut out the gene insert from gel

Method: Use a UV light to visualize the band and cut out with scalpel. Use the gel isolation kit from Cytiva.

Protocol:

• Stain gel in Ethidium bromide for 15 min.
• Take gel to UV light and visualize it.
• Take scalpel and cut out the fragment from gel
• Follow protocol from the gel isolation kit.

Result: Concentration for copA was 65 ng/μl and for napA it was 11.8 ng/μl.

1. Ligation & transformation

Date: 05.08-06.08 & 08.08-09.08

Duration: overnight

Goal: ligate the insert into pet16b vector

Method: Use a T4 ligase and T4 buffer. Then do transformation into E. coli BL21

Protocol:

• The online protocol for ligation from NEB was used, as well as the online calculator from NEB.
• Transformation

Results: All the plates showed colonies.

1. Diagnostic restriction digestion

Date: 09.08-10.08

Duration: 1 day

Goal: Check if the correct plasmid is present in BL21.

Method: Use plasmid isolation kit from Qiagen and then do restriction digest diagnosis

Hypothesis: See 2 bands on gel indication that the insert is present.

Protocols:

• Inoculate culture overnight
• Isolate plasmid by using the protocol from the plasmid isolation kit
• Restriction digest

Results:

Fig. 26 | Gel electrophoresis for copA, and napA digested with NdeI and XbaI. A) Digested napA. B) Digested copA.

Conclusion: For napA, two of the samples seemed to have the insert present. For copA it looked like the inserts were not in there.

5. Sequencing

Date: 31.08

Duration: preparation: 20min. The sequencing at the company: 1-2 days

Goal: Get confirmation that the inserts are present

Method: Sanger sequencing

Hypothesis: The results from sequencing should align with the gene fragments that were inserted into the plasmid. Sequencing was done for cup1, copA, and napA

Protocol: Add 5 μl of DNA, 2μl of one primer (forward or reverse) into an Eppendorf tube, bring volume up to 20 μl.

Results:

Fig. 27 | Schematic representation of sequence alignment. Sequence is aligned to cup1.

Fig. 28 | Schematic representation of sequence alignment. Arrow is not colored in, indicating no alignment with copA.

Fig. 29 | Schematic representation of sequence alignment. Arrow is not colored in, indicating no alignment with napA.

References:

1. Gurunathan, S. et al. Biosynthesis, purification and characterization of silver nanoparticles using Escherichia coli. Colloids Surf B Biointerfaces 74, 328–335 (2009).
2. Cheng, L. C. et al. Seedless, silver-induced synthesis of star-shaped gold/silver bimetallic nanoparticles as high efficiency photothermal therapy reagent. J Mater Chem 22, 2244–2253 (2012)
3. Gou, Y., Zhou, R., Ye, X., Gao, S., & Li, X. (2015). Highly efficient in vitro biosynthesis of silver nanoparticles using Lysinibacillus sphaericus MR-1 and their characterization. Science and Technology of Advanced Materials, 16(1), 015004. https://doi.org/10.1088/1468-6996/16/1/015004
4. Kumar, S. A. et al. Nitrate reductase-mediated synthesis of silver nanoparticles from AgNO 3. Biotechnol Lett 29, 439–445 (2007).

Supplementary experiments:

1. Calibration of absorbance measurements between a 96 well plate and spectrophotometer

Date: 20.07

Duration: 2h + overnight culture

Goal: Make a correlation for absorbance measurements between a plate reader and a spectrophotometer, to avoid discrepancies.

Method: measure absorbance of a dilution range of a liquid culture of WT E. coli BL21 and measure on the two machines.

Protocol: Calibration of Absorbance Measurement Between a 96-Well Plate and Spectrophotometer

Results:

Fig. 30 | Absorbance measurements of calibration between the spectrophotometer and platereader.

Raw Absorbance Data

Conclusion: the difference in absorbance is insignificant, so no cross calibration is necessary for all experiments.

2. Comparing nanoparticle production in an eppendorf tube, in a flask, and in a 24-well-plate

Date: 14.08-16.08

Duration: 1.5 days

Goal: Identify which platform: eppendorf tube, 50mL flask or a 24-well plate, is the most suitable for nanoparticle production.

Method: Compare absorption spectra of nanoparticle synthesis using supernatant of WT E. coli BL21 grown in MH, when synthesis happens in an eppendorf tube, a 50mL flask and a 24-well plate.

Hypothesis: Shaking in a flask is more intense and probably results in a better distribution of salts, resulting in a more uniform size distribution of nanoparticles. According to this logic, absorbance intensity would be as follows: flask > plate > eppendorf tube.

Protocol: Compare Nanoparticle Synthesis in an Eppendorf Tube vs, Flask vs. Well Plate

Notes: We had to wait one day due to the culture not being ready yet. The LB-nitrate medium and ddH₂O samples were prepared in advance and stored in the fridge overnight

Results:

Fig. 31 | Absorbance measurements of biologically produced nanoparticles using wildtype Escherichia coli BL21 in different platforms: Eppendorf tubes, flasks, and plate.

Raw Absorbance Data

Conclusion: As expected, conducting nanoparticle synthesis in a flask yields the highest absorbance.

Discussion: The discrepancy between the flask and the eppendorf tube for supernatant and media samples cannot easily be explained with the existing data, so more experiments have to be conducted in that regard. We also set up two other experiments that looked into nanoparticle production in bigger volumes and the influence of shaking speeds on that.

3. Obtaining a correlation between nanoparticle concentration and absorbance.

Date: 15.08

Duration: 1h

Goal: show that a higher absorbance value corresponds with a higher concentration of nanoparticles.

Method: chemically produce a dispersion of nanoparticles using ascorbic acid (use the 1.85:1 ratio). Next, make absorbance measurements for a dilution series of 1x, 2x, 4x, 8x, 16x, 32x.

Hypothesis: a correlation of the format: A = A₀*(½)ⁿ is expected, which would mean that there is a linear correlation between nanoparticle concentration and absorbance.

Protocol: Correlating Nanoparticle Concentration and Absorbance

Results:

Fig. 32 | Absorbance measurements of chemical produced nanoparticles using ascorbic acid with different dilutions to test the influence on absorbance.

Fig. 33 | Linear correlation between nanoparticle concentration and absorbance.

Raw Absorbance Data

Conclusion: higher absorbance corresponds with a higher concentration of nanoparticles. Therefore, in most experiments we equate a higher absorbance to a higher concentration of nanoparticles at a specific wavelength (typically 800 nm).

Discussion: this might only be true for nanoparticles from the same sample, as they maintain the same shape & size. However, this doesn't mean that it's true for samples in which nanoparticles are different in size & shape. Namely, the same absorbance can be achieved with individual nanoparticles and, accidentally, with agglomerates of nanoparticles.

4. Obtaining absorption spectra of HAuCl₄ and AgNO₃ in ddH2O

Date: 16.08-17.08

Duration: 24h

Goal: show that the shape of the spectra in experiments with reducing agents is not caused by oxidation of silver/gold or the formation of AgCl

Method: observe spectra of silver, gold, gold & silver in DI water at t=0, t=1d and t=9d.

Hypothesis:

• AgNO3 is expected to give a peak at 325 nm¹
• HAuCl4 is expected to give a peak at 225 nm and 310 nm²
• AgCl is expected to give a peak in the 200-300 nm region³
• Ag2O is expected to give a peak at 450 nm⁴
• Au doesn't oxidize, so no gold oxides will be formed.

Protocol: Absorption Spectra of Salts in ddH2O Water

Results:

Fig. 34 | Absorbance measurements of ddH2O with silver and gold salts after 0h.

Fig. 35 | Absorbance measurements of ddH2O with silver and gold salts after 24h.

Raw Absorbance Data

Conclusion: compounds present in sufficient concentrations to be observed in a change of absorbance are mostly situated in the <400 nm region, which corresponds to the predicted positions of the compound absorbance peaks. Therefore, the peaks >400 nm observed in all other experiments are not caused by salts, secondary reactions or oxidation of salts.

5. Recording dynamic absorbance measurements of nanoparticle synthesis using ascorbic acid

Date: 19.08

Duration: 1h

Goal: Record the shifting of the peak to the left with time for nanoparticle synthesis using ascorbic acid.

Method: Make nanoparticle synthesis samples in a 24-well plate and measure the absorption spectrum of the samples every 2-3 minutes.

Hypothesis: a shift towards 600 nm is expected, as this is the wavelength, at which peaks are observed when measuring biologically-synthesized nanoparticle samples after 24h.

Protocol: Ascorbic Acid Chemical Synthesis Dynamic Measurements

Notes:

• The time to scan absorbance of 1 well is ~3s.
• Time between the addition of ascorbic acid to the first well and before the first measurement was 3min 5s.
• The plate was shaken for 15s with 810rpm and 2cm amplitude between measurements

Results:

Fig. 36 | Dynamic absorbance measurements of chemically produced nanoparticles using ascorbic acid for a total duration of 1h.

Raw Absorbance Data (t = 1 h, measurements every 2-3 minutes)

Raw Absorbance Data (t = 24 h, measurements every ~15 minutes)

Conclusion: The peak indeed shifts towards 625 nm as the time progresses. The shifting stabilizes after approximately one hour.

Discussion: Such a result further emphasizes the necessity for using surfactants or capping compounds, so as to prevent agglomeration and collapsing of the nanoparticles and the subsequent shifting of the peak.

6. Comparing different techniques for obtaining a cell-free system

Date: 18.08-20.08

Duration: 2 days

Goal: identify the most reliable method for obtaining a cell-free system.

Method: Add Kanamycin/Chloramphenicol to the supernatant or filter-sterilize it and plate out overnight. Check if growth can be observed.

Hypothesis: antibiotics will not eliminate all possibilities for bacterial growth, so only sterile filtering will fully eliminate growth.

Protocol: Filter Sterilization vs. Antibiotic Treatment

Results:

Fig. 37 | Agar plates with bacteria treated with either kanamycin, ampicillin or chloramphenicol

Only on the filter-sterilized plate no growth is observed.

Conclusion: proceed in all experiments with filtered supernatant

7. Comparing protein expression of WT E. coli BL21 in media used for nanoparticle production

Date: 16.08-17.08

Duration: 1.5 days

Goal: Study if growth in a particular medium results in an overexpression of similar genes, possibly the same genes that we are overexpressing by induction.

Method: Grow a WT E Coli BL21 culture in one of the media for 24h, take a sample and prepare for SDS.

Hypothesis: MH Broth and LB-Nitrate were expected to yield thicker bands for some proteins that would be responsible for mitigating the effects of the nature of these broths.

Protocol: Comparing Protein Expression of Wild Type Escherichia coli BL21 in Media Used for Nanoparticle Production

Results:

Fig. 38 | SDS gel showing BL21 grown in either MHB, MHB with nitrate, LB low salt, LB, or LB with nitrate.

Conclusion:

• MHB and MHB-Nitrate give very similar bands that are different than LB control
• LB Low salt also gives a different band than the LB control
• LB-Nitrate gives almost identical bands as LB.

8. Testing the influence of ingredients of different media on nanoparticle synthesis

Date: 16.08-17.08

Duration: 1.5 days

Goal: show which components of the different media are responsible for nanoparticle formation.

Method: measure absorbance of aqueous solutions of individual ingredients of LB, TSBS & MH and metal salts at t=0 and at t=24h.

Hypothesis: tryptone, beef infusion and yeast extract all contain some form of proteins and are, therefore, expected to contain reducing agents. With this, they are expected to cause nanoparticle formation.

Protocol: Checking The Influence of Media Compounds on Nanoparticle Synthesis

Notes: water blanks were done with ddH₂O instead of DI water, because sterile DI water was not available.

Results:

Fig. 39 | Absorbance measurements of samples with LB medium ingredients in DI Water, with HAuCl₄ and AgNO₃ after 24h of incubation at room temperature. Used conditions: [HAuCl₄] = 3 mM, [AgNO₃] = 0.3 mM. LB composition according to Green & Sambrook¹

Fig. 40 | Absorbance measurements of samples with MH medium ingredients in DI Water, with HAuCl₄ and AgNO₃ after 24h of incubation at room temperature.

Used conditions: [HAuCl₄] = 3 mM, [AgNO₃] = 0.3 mM. MH composition according to the recipe obtained from Sigma-Aldrich² with a replacement of Casein Hydrolysate by Tryptone

Fig. 41 | Absorbance measurements of samples with TSBS medium ingredients in DI Water, with HAuCl₄ and AgNO₃ after 24h of incubation at room temperature.
Used conditions: [HAuCl₄] = 3 mM, [AgNO₃] = 0.3 mM. TSBS composition according to Center for Food Safety and Applied Nutrition³ with a replacement of Soy Extract by Yeast Extract.

Raw Absorbance Data

Conclusion: tryptone, yeast extract and beef infusion indeed have a reducing capacity and result in nanoparticle formation.

Discussion: The pre-made formulations of the broths give higher absorbance values than tryptone and other individual components. This means that there is potentially some interaction between the constituents of the media that results in a higher reducing capacity. This is further tested for MH in the next described experiment.

9. Testing if interaction between different MH Broth ingredients has influence on nanoparticle synthesis

Date: 24.08-25.08

Duration: 1.5 days

Goal: for the selected optimal broth, show which components interact positively, resulting in a higher reducing capacity

Method: for tryptone, beef infusion and starch, show the influence of all possible combinations on nanoparticle formation.

Hypothesis: tryptone and beef infusion have a positive interaction, since they both contain proteins and, most probably, reducing agents.

Protocol: Checking The Interaction Between Different Mueller Hinton Broth Components on Nanoparticle Synthesis

Notes: water blanks were done with ddH₂O instead of DI water, because sterile DI water was not available.

Results:

Fig. 42 | Absorbance measurements to assess if different components of Mueller Hinton broth have influence on nanoparticle synthesis.

Raw Absorbance Data

Conclusion:

• MH Broth performs better than MH Supernatant and than all combinations of ingredients separately
• The BI + Tryptone + Starch absorbance being lower than the pre-made MH is probably due to the fact that the BI that is available at our lab is different from the one they used for the pre-made powder.

10. Interlab measurements

Date: 25.07-05.08

Duration: 7 days (+ pre-cultured transformations)

Goal: Participate in the Interlab study to contribute to the reproducibility and reliability of fluorescence measurements in different labs all around the globe

Method: iGEM website with description of the protocols for three experiments

Notes:

• 25.07: execute transformations on plates
• 27.07: check growth on plates
  • Not everywhere ⇒ wait for another day
• 28.07: check growth on plates + make liquid cultures
• 29.07: stop the experiment, because plate reader wouldn’t be available
  • Make sure that all the transformants are still available
  • Plates with E1, E2, P1 and P2 did not contain any transformants, so these samples were excluded from the recorded results in the subsequent measurements.
• 30.07: prepare for experiment + perform calibration
  • Calibration performed
  • plate out 50uL of liquid cultures for H1, H2, L1, L2 as not enough growth was obtained on the plates
  • 4 plates with LB-agar 34 ug/ml Chloramphenicol
• 01.08: the plates from 29.07 had overgrown, so they were plated out again
  • 4 plates with LB-agar 34 ug/ml Chloramphenicol
• 03.08: Liquid cultures
  • Make liquid cultures of the 48 plates
  • 12 mL per culture
  • 576 mL LB necessary
  • Cultures in shaker at 13:20
• 04.08: Wasted dilutions
  • 09:10 Cultures out of incubator, made 1:10 dilutions
  • PLATE READER UNAVAILABLE SO MEASUREMENTS TOMORROW
  • 12:15 1:10 dilutions in shaker
• 05.08: Final measurements:
  • Deviation from protocol: no 1:10 dilution. We immediately dilute 1:3 in cuvettes
  • Deviation: Experiment 1 Plate 1
    • Column 6 (Test Device 3) was not pipetted due to the failed transformation (E1&E2)
    • Column 8 (Test Device 5) was put into column 11 instead due to a pipetting mistake. But, the data was submitted according to the layout provided in the protocol.
  • Deviation: Experiment 2 Plate 2
    • Column 9 (Test Device 5) was not pipetted due to the failed transformation (P1&P2)
  • 12:30 back dilution cultures in shaker 37°C
  • 13:13 experiment 3 plate 1 in shaker 37°C
  • 16:45 everything out of shaker
    • A1 possibly contaminated. Cap was off falcon tube in shaker
  • Data for the final measurements was recorded at different gain values to prevent overexposure, which complicated the correlation of data on the side of iGEM

General: plates were not held on ice while performing pipetting

Results:

• Raw Data
• Submitted Data

Discussion: Unfortunately, this year’s Interlab was affected by a manufacturing issue. The silica nanobeads, which were purposed for calibration, gave no signal during the calibration step. This affected our calibration as well, which means that our measurements would have to be assessed by quality control first, after which their usability will be evaluated.

References:

1. Green, R. M. & Sambrook, J. Molecular Cloning This is a free sample of content from Molecular Cloning: A Laboratory Manual, 4th edition. Click here for more information or to buy the book. www.cshlpress.org (2012).
2. Sigma-Aldrich. (2018). 70192 Mueller Hinton Broth (M-H Broth). Retrieved September 21, 2022
3. Center for Food Safety and Applied Nutrition. (2017, October 24). BAM Media M154: Trypticase (Tryptic) Soy Broth. Retrieved September 21, 2022, from https://www.fda.gov/food/laboratory-methods-food/bam-media-m154-trypticas

Implementation experiments:

1. Testing nanoparticle production using a bioreducing system in a bigger volume

Date: 26.08-27.08

Duration: 1.5 days

Goal: Scale-up the nanoparticle production to a bigger volume.

Method: Test if straightforward scale-up of the nanoparticle production process is possible by testing the same conditions as in a 24-well plate in a 50mL flask.

Hypothesis: With the same conditions, we expected that the produced nanoparticles would have similar properties (absorbance, size, morphology).

Protocol: Big Batch Experiment

Results:

TEM Pictures

Conclusion: Our hypothesis was proven wrong. As seen in the TEM Pictures, the sample contained more agglomerates, while no nanoparticles with a similar branched morphology were observed.

Discussion: scale-up of the production process demands fine-tuning of the parameters that influence nanoparticle synthesis. We expect one such property to be the shaking speed, or centrifugal force, that the reaction mix experiences. This could have an influence on the clumping of nanoparticles and the uniformity of the distribution of the dissolved compounds in solution. In order to test the former parameter, the next described experiment was set up.

2. Testing the influence of shaking speed on nanoparticle production using a bioreducing system

Date: 26.08-29.08

Duration: 3.5 days

Goal: identify if the shaking speed and the centrifugal force that the reaction liquid experiences has an effect on the quality of the produced nanoparticles.

Method: set up shaking flasks with silver and gold and WT + ASKA strain supernatants. The shaking settings are 0/20/60rpm.

Hypothesis: intense shaking “presses” the smaller particles together, resulting in more agglomeration. And no shaking at all will lower the stochastic interactions necessary for NP formation, resulting in less developed NPs.

Protocol: Rocking Speed Experiment

Results:

• Absorbance:
  • Shows no significant difference between different shaking settings
  • Shows a significant difference between WT and ASKA strains

Fig. 43 | Absorbance graphs for E.coli BL21, ASKA-MalE, ASKA-NapA, and ASKA-CueO. A) Absorption spectrum of E.coli BL21 at three different shaking speeds 0 rpm, 20 rpm, and 60 rpm. B-D) Absorption spectrum of ASKA-MalE, ASKA-NapA, and ASKA-CueO respectively at three different shaking speeds 0 rpm, 20 rpm, and 60 rpm.

Raw Absorbance Data

• TEM:
  • Less agglomeration is observed for 60rpm than for 0rpm
  • NapA seems to have the most positive effect on nanoparticle formation.
  • The NapA 60-speed seems to have more developed nanoparticles than the 0-speed NapA.

TEM Pictures

Conclusion: Shaking has a positive effect on nanoparticle formation in preventing agglomeration and in the secondary structure development of nanoparticles.

• Overexpression of ASKA genes has a positive effect on nanoparticle formation, especially napA

3. Proof of concept: light into heat conversion using biologically-produced nanoparticles

Date: 08.09-09.09

Duration: 2 days

Goal: Show that the presence of our nanoparticles in bulk results in an increase of temperature when a laser at 800 nm is shone at it.

Method: Disperse extracted nanoparticles into 3 mL of ddH₂O, place a temperature probe in the corner of a cuvette. Shine a laser for 5 min, while recording the change in temperature. Control with ddH₂O + AgNO3 + HAuCl4.

Hypothesis: our extracted nanoparticles show a non-zero absorbance at 800 nm, so there are nanoparticles that will absorb light of that wavelength and convert it into heat. A significant temperature change in comparison to control is expected.

Protocol: See the Proof of Concept page for a detailed description of the set up, the results and the implication of those results for the scope of our project.

Proof of Concept

Results:

Fig. 44 | Absorbance measurements of biologically produced nanoparticles using wildtype Escherichia coli BL21 supernatant and lystate in combination with silver and gold salts, ratio 1.85:1.

Fig. 45 | Heat experiments of biologically produced nanoparticles exposed to a near-infrared laser for five minutes.

Raw Absorbance Data

Raw Heat Experiment Data

TEM Pictures

Conclusion: The sample containing biologically-produced nanoparticles resulted in a significant increase in temperature, as compared to the control. This means that the nanoparticles produced with Binanox have the capacity to heat up and cause cell death in a cell line.

4. Extraction of nanoparticles by size using an ultracentrifuge

Date: 17.08-18.08

Duration: 2 days

Goal: obtain a purified fraction of nanoparticles with a peak absorption at 800 nm.

Method: Place a mixed nanoparticle sample on a sucrose gradient in an ultracentrifuge to obtain layer separation of nanoparticles by size.

Hypothesis: nanoparticles of a particular size are expected to have the desired absorbance at 800 nm. This is the layer that we want to isolate by ultracentrifugation.

Protocol: Ultracentrifuge

Results:

Fig. 46 | Absorption spectra of the different isolated layers, denoted by the respective sucrose concentration in g/L.

Discussion: during the ultracentrifugation process a crucial mistake was made, we did not make use of a ‘swing bucket’. Due to using normal ultracentrifugation tubes the sample was pressed to the side of the tube and made a smear here. Because the sample was smudged to the side of the sample it did not go properly through the sucrose gradient layers. The fractions that were extracted we scraped from the side and are not properly seperated. This is why the peaks are at the same absorbance.

Conclusion: No conclusive extraction results due to the wrong type of ultracentrifugation extraction. The use of a swing bucket system is crucial for the sucrose gradient extraction method to work.