iGEM 2022 — Team leiden

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
{% block page\_content %}

# {{ self.title() }}

An organized overview of all experiments conducted in the six weeks of lab time, aimed at exploration and optimization of the Binanox microbial factory

## PRELIMINARY

Experiments aimed at exploration and identification of feasible ideas, which were employed in the subsequent experiments

## CORE

Experiments aimed at optimization of PTT-optimized nanoparticle production using Binanox

## SUPPLEMENTARY

Experiments aimed at elucidating certain aspects of nanoparticle production using bioreduction

## IMPLEMENTATION

Experiments aimed at testing the scale-up opportunities of Binanox, as well as proof of concept

* CONTENTS
* [Preliminary](#preliminary)
* [Core](#core)
* [Supplementary](#supplementary)
* [Implementation](#implementation)

# 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](https://2022.igem.wiki/leiden/protocols#protocol-p1)

**Results**: [Raw Absorbance Data](https://doi.org/10.5281/zenodo.7181138), [TEM pictures](https://doi.org/10.5281/zenodo.7181064)

**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](https://2022.igem.wiki/leiden/protocols#protocol-p2)* [Continuation of Preliminary Flask Experiment and Setting up Secondary Experiments](https://2022.igem.wiki/leiden/protocols#protocol-p3)

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

**Results**

[Raw Absorbance Data](https://doi.org/10.5281/zenodo.7181177)

![alt_text](https://static.igem.wiki/teams/4259/wiki/notebook/fig1.png "image_tooltip")

**Fig. 1 |** Absorbance measurements of wildtype E.coli BL21 in combination with 1mM HAuCl4 and 1 mM AgNO3. After 1, 2, and 3 days with two measurements per day.

![alt_text](https://static.igem.wiki/teams/4259/wiki/notebook/fig2.png "image_tooltip")

**Fig. 2 |** Absorbance measurements of wildtype E.coli BL21 in combination with 10mM HAuCl4 and 10 mM AgNO3. 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](https://2022.igem.wiki/leiden/protocols#protocol-p4)

**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)

![alt_text](https://static.igem.wiki/teams/4259/wiki/notebook/figure-3-plates.png "image_tooltip")

**Fig. 3** | Plates showing bacterial growth after sonication

**Results**:

[Raw Absorbance Data](https://doi.org/10.5281/zenodo.7181193)

![alt_text](https://static.igem.wiki/teams/4259/wiki/notebook/fig4.png "image_tooltip")

**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.

![alt_text](https://static.igem.wiki/teams/4259/wiki/notebook/fig5.png "image_tooltip")

**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.

![alt_text](https://static.igem.wiki/teams/4259/wiki/notebook/fig6.png "image_tooltip")

**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.

![alt_text](https://static.igem.wiki/teams/4259/wiki/notebook/fig7.png "image_tooltip")

**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](https://2022.igem.wiki/leiden/protocols#protocol-p5)

**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](https://doi.org/10.5281/zenodo.7181221)

![alt_text](https://static.igem.wiki/teams/4259/wiki/notebook/fig8.png "image_tooltip")

**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.

![alt_text](https://static.igem.wiki/teams/4259/wiki/notebook/fig9.png "image_tooltip")

**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.

![alt_text](https://static.igem.wiki/teams/4259/wiki/notebook/fig10.png "image_tooltip")

**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.

![alt_text](https://static.igem.wiki/teams/4259/wiki/notebook/fig11.png "image_tooltip")

**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](https://2022.igem.wiki/leiden/protocols#protocol-p6)

**Notes:**

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

**Results**:

![alt_text](https://static.igem.wiki/teams/4259/wiki/notebook/fig12.png "image_tooltip")

**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.

![alt_text](https://static.igem.wiki/teams/4259/wiki/notebook/fig13.png "image_tooltip")

**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](https://doi.org/10.5281/zenodo.7181245)

**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](https://2022.igem.wiki/leiden/protocols#protocol-p7)

**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**:

![alt_text](https://static.igem.wiki/teams/4259/wiki/notebook/fig14.png "image_tooltip")

**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.

![alt_text](https://static.igem.wiki/teams/4259/wiki/notebook/fig15.png "image_tooltip")

**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](https://doi.org/10.5281/zenodo.7181275)

[Raw Absorbance Data](https://doi.org/10.5281/zenodo.7181282)

**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](https://2022.igem.wiki/leiden/protocols#protocol-p8)

**Results**:

![alt_text](https://static.igem.wiki/teams/4259/wiki/notebook/fig16.png "image_tooltip")

**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](https://2022.igem.wiki/leiden/protocols#protocol-p9)* [Luminescence experiment](https://2022.igem.wiki/leiden/protocols#protocol-p10)

**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](https://doi.org/10.5281/zenodo.7181288)

![alt_text](https://static.igem.wiki/teams/4259/wiki/notebook/fig17.png "image_tooltip")

**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).- 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](https://2022.igem.wiki/leiden/protocols#protocol-c1)* [Compare Supernatants and Lysates of 4 Different Broths](https://2022.igem.wiki/leiden/protocols#protocol-c2)

**Results:**

![alt_text](https://static.igem.wiki/teams/4259/wiki/notebook/fig18.png "image_tooltip")

**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.

![alt_text](https://static.igem.wiki/teams/4259/wiki/notebook/picture6.png "image_tooltip")

**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](https://doi.org/10.5281/zenodo.7181325)

[TEM Pictures](https://doi.org/10.5281/zenodo.7181321)

**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](https://2022.igem.wiki/leiden/protocols#protocol-c3)

**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**:

![alt_text](https://static.igem.wiki/teams/4259/wiki/notebook/fig20a.png "image_tooltip")
![alt_text](https://static.igem.wiki/teams/4259/wiki/notebook/fig20b.png "image_tooltip")
![alt_text](https://static.igem.wiki/teams/4259/wiki/notebook/fig20c.png "image_tooltip")

**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](https://doi.org/10.5281/zenodo.7181368)

[TEM Pictures](https://doi.org/10.5281/zenodo.7181376)

**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](https://2022.igem.wiki/leiden/protocols#protocol-c4)

**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**:

![alt_text](https://static.igem.wiki/teams/4259/wiki/notebook/fig21.png "image_tooltip")

**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 A800 nm for the same measurements. Error bars represent standard error (n = 3).

[Raw Absorbance Data](https://doi.org/10.5281/zenodo.7181380)

**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](https://2022.igem.wiki/leiden/protocols#protocol-c5)

**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](https://doi.org/10.5281/zenodo.7181389)

**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](https://2022.igem.wiki/leiden/protocols#protocol-c6)

**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**:

![alt_text](https://static.igem.wiki/teams/4259/wiki/notebook/fig22.png "image_tooltip")

**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.

![alt_text](https://static.igem.wiki/teams/4259/wiki/notebook/fig23.png "image_tooltip")

**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](https://doi.org/10.5281/zenodo.7181413)

[Raw Absorbance Data](https://doi.org/10.5281/zenodo.7181405)

**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 nanoparticles1,4

**Protocol: [Compare A/K/T pellet lysate with supernatant of WT grown on MH-nitrate](https://2022.igem.wiki/leiden/protocols#protocol-c7)**

**Results**: results can be found on the results page.

[Raw Absorbance Data](https://doi.org/10.5281/zenodo.7181421)

[Statistical Analysis](https://doi.org/10.5281/zenodo.7181423)

**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](https://2022.igem.wiki/leiden/protocols#protocol-c8)**

**Results**:

![alt_text](https://static.igem.wiki/teams/4259/wiki/notebook/sds-1.png "image_tooltip")

**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](https://2022.igem.wiki/leiden/protocols#protocol-c9)
* [Transformation](https://2022.igem.wiki/leiden/protocols#protocol-c10)

**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](https://2022.igem.wiki/leiden/protocols#protocol-c11)

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

**Results:**

![alt_text](https://static.igem.wiki/teams/4259/wiki/notebook/figure-25.png "image_tooltip")

**Fig. 25 |** Gel electrophoresis for *cup1, copA, and napA* digested with *Nde*I and *Xba*I. 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](https://2022.igem.wiki/leiden/protocols#protocol-c10)

**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](https://2022.igem.wiki/leiden/protocols#protocol-c11)

**Results:**

![alt_text](https://static.igem.wiki/teams/4259/wiki/notebook/figure-26-gel-copa-napa-jpg.png "image_tooltip")

**Fig. 26 |** Gel electrophoresis for *copA, and napA* digested with *Nde*I and *Xba*I. 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:**

![alt_text](https://static.igem.wiki/teams/4259/wiki/notebook/figure-27-mt-sequencing-results.png "image_tooltip")

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

![alt_text](https://static.igem.wiki/teams/4259/wiki/notebook/figure-28-copa-sequencing-results.png "image_tooltip")

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

![alt_text](https://static.igem.wiki/teams/4259/wiki/notebook/figure-29-sequencing-results-napa.png "image_tooltip")

**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).- 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)- 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- 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](https://2022.igem.wiki/leiden/protocols#protocol-s1)**

**Results**:

![alt_text](https://static.igem.wiki/teams/4259/wiki/notebook/fig26.png "image_tooltip")

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

[Raw Absorbance Data](https://doi.org/10.5281/zenodo.7181441)

**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](https://2022.igem.wiki/leiden/protocols#protocol-s2)

**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**:

![alt_text](https://static.igem.wiki/teams/4259/wiki/notebook/fig27.png "image_tooltip")

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

[Raw Absorbance Data](https://doi.org/10.5281/zenodo.7181449)

**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](https://2022.igem.wiki/leiden/protocols#protocol-s3)**

**Results**:

![alt_text](https://static.igem.wiki/teams/4259/wiki/notebook/fig28.png "image_tooltip")

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

![alt_text](https://static.igem.wiki/teams/4259/wiki/notebook/fig29.png "image_tooltip")

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

[Raw Absorbance Data](https://doi.org/10.5281/zenodo.7181453)

**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](https://2022.igem.wiki/leiden/protocols#protocol-s5)**

**Results**:

![alt_text](https://static.igem.wiki/teams/4259/wiki/notebook/fig30.png "image_tooltip")

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

![alt_text](https://static.igem.wiki/teams/4259/wiki/notebook/fig31.png "image_tooltip")

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

[Raw Absorbance Data](https://doi.org/10.5281/zenodo.7181459)

**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](https://2022.igem.wiki/leiden/protocols#protocol-s6)

**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**:

![alt_text](https://static.igem.wiki/teams/4259/wiki/notebook/fig32.png "image_tooltip")

**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)](https://doi.org/10.5281/zenodo.7181464)

[Raw Absorbance Data (t = 24 h, measurements every ~15 minutes)](https://doi.org/10.5281/zenodo.7181470)

**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](https://2022.igem.wiki/leiden/protocols#protocol-s7)**

**Results**:

![alt_text](https://static.igem.wiki/teams/4259/wiki/notebook/figure-37-plates-antibiotics.png "image_tooltip")

**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](https://2022.igem.wiki/leiden/protocols#protocol-s8)**

**Results**:

![alt_text](https://static.igem.wiki/teams/4259/wiki/notebook/figure-38-sds-2.jpg "image_tooltip")

**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](https://2022.igem.wiki/leiden/protocols#protocol-s9)**

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

**Results**:

![alt_text](https://static.igem.wiki/teams/4259/wiki/notebook/fig35.png "image_tooltip")

**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

![alt_text](https://static.igem.wiki/teams/4259/wiki/notebook/fig36.png "image_tooltip")

**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

![alt_text](https://static.igem.wiki/teams/4259/wiki/notebook/fig37.png "image_tooltip")

**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](https://doi.org/10.5281/zenodo.7181480)

**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](https://2022.igem.wiki/leiden/protocols#protocol-s10)

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

**Results**:

![alt_text](https://static.igem.wiki/teams/4259/wiki/notebook/fig38.png "image_tooltip")

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

[Raw Absorbance Data](https://doi.org/10.5281/zenodo.7181484)

**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](https://technology.igem.org/interlabs/2022) 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](https://doi.org/10.5281/zenodo.7181490)* [Submitted Data](https://doi.org/10.5281/zenodo.7181493)

**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).- Sigma-Aldrich. (2018). 70192 Mueller Hinton Broth (M-H Broth). Retrieved September 21, 2022- 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](https://2022.igem.wiki/leiden/protocols#protocol-i1)**

**Results**:

[TEM Pictures](https://doi.org/10.5281/zenodo.7181504)

**Conclusion:** Our hypothesis was proven wrong. As seen in the [TEM Pictures](https://doi.org/10.5281/zenodo.7181504), 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](https://2022.igem.wiki/leiden/protocols#protocol-i2)**

**Results**:

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

![alt_text](https://static.igem.wiki/teams/4259/wiki/notebook/fig39.png "image_tooltip")

**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](https://doi.org/10.5281/zenodo.7181510)

* 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](https://doi.org/10.5281/zenodo.7181528)

**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](https://2022.igem.wiki/leiden/protocols#protocol-i3)

**Results**:

![alt_text](https://static.igem.wiki/teams/4259/wiki/notebook/fig40.png "image_tooltip")

**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.

![alt_text](https://static.igem.wiki/teams/4259/wiki/notebook/fig41.png "image_tooltip")

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

[Raw Absorbance Data](https://doi.org/10.5281/zenodo.7181550)

[Raw Heat Experiment Data](https://doi.org/10.5281/zenodo.7184953)

[TEM Pictures](https://doi.org/10.5281/zenodo.7181558)

**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](https://2022.igem.wiki/leiden/protocols#protocol-i4)

**Results**:

![alt_text](https://static.igem.wiki/teams/4259/wiki/notebook/fig42.png "image_tooltip")

**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.

## References:

1. Nakamura, T., Magara, H., Herbani, Y. & Sato, S. Fabrication of silver nanoparticles by highly intense laser irradiation of aqueous solution. *Appl Phys A Mater Sci Process* **104**, 1021–1024 (2011).- King, S. R., Massicot, J. & McDonagh, A. M. A Straightforward Route to Tetrachloroauric Acid from Gold Metal and Molecular Chlorine for Nanoparticle Synthesis. *Metals (Basel)* **5**, 1454–1461 (2015).- Trinh, D., Nguyen, B. & Nguyen, T. Preparation and characterization of silver chloride nanoparticles as an antibacterial agent. *Advances in Natural Sciences: Nanoscience and Nanotechnology* **6**, 45011 (2015).- Salman Elfadel Tyfor, M. Study of Some optical properties of silver oxide ( o Ag 2 ) using UV-Visible spectrophotometer. *IOSR Journal of Applied Physics (IOSR-JAP* **8**, 51–54.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block page\_content %}

# {{ self.title() }}

Iterative optimization of a scalable biomanufacturing process for photothermal therapy-optimized nanoparticle production

## CYCLE №1

Identify the optimal ratio of gold:silver salts for synthesis of photothermal therapy (PTT)-optimized nanoparticles

## CYCLE №2

Identify the growth medium for obtaining the optimal bioreducing mix for the production of PTT-optimized nanoparticles

## CYCLE №3

Test the influence of Escherichia coli genes copA, napA, cueO, and melA on nanoparticle synthesis

## CYCLE №4

Test if growth on a nitrate-containing medium benefits the bioreducing capacity of the supernatant used for nanoparticle production

* CONTENTS
* [Overview](#overview)
* [Introduction](#introduction)
* [Cycle 1](#cycle-1)
* [Cycle 2](#cycle-2)
* [Cycle 3](#cycle-3)
* [Cycle 4](#cycle-4)
* [Conclusion](#conclusion)
* [Future experiments](#future)
* [References](#references)

# Overview

With Binanox, we set ourselves the goal of establishing a novel, scalable process for the production of bimetallic nanoparticles using a cell-free system with metal-reducing proteins. Within the scope of our project, we approach process design from a scale-up perspective by focusing on understanding and optimizing the process at a lab scale first, then proceeding to pilot and factory levels. Therefore, we employed the Design-Build-Test-Learn engineering cycle framework to ensure that our system is optimized at every iteration. On this page, you will find a description of how four such cycles guided the creation of Binanox.

![](https://static.igem.wiki/teams/4259/wiki/engineering-page/fig1.png)

**Fig. 1** | Design-Build-Test-Learn cycle

![Overview of engineering cycles](https://static.igem.wiki/teams/4259/wiki/engineering-page/fig2.png)

# Introduction

To approach process design efficiently, we started by looking at the monumental efforts of our predecessors1. Due to similarities in reaction conditions, we took green chemical production methods as a reference point for our process design. Namely, the synthesis of nanoparticles with ascorbic acid, as outlined by Cheng et al. 2 was deemed the most representative of our aspirations. The authors obtained star-shaped nanoparticles with a plasmon peakinfo at 800 nm. This fits almost all our prerequisites (read more about this on the [Project Description](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27description%27%29%20%7D%7D) page), as these properties are optimal for use in photothermal therapy (PTT). However, this methodology does not yield bimetallic nanoparticles. We aimed to make this possible with our biomanufacturing process by using this synthesis methodology as the starting point for our process design.

Ascorbic acid is among the standard methodologies for the production of nanoparticles, but it poses several difficulties in scaling the production industrially3. At the same time, the most common chemical reducing agent for industrial production is sodium borohydride, which produces toxic, pyrophoric and flammable compounds upon oxidation (B2H6 and H2 gasses)4. Using biological reducing agents, we want to provide a safe, scalable, and efficient methodology for producing bimetallic star-shaped nanoparticles5.

# Cycle 1. Determining the optimal HAuCl4:AgNO3 ratio for PTT-optimized nanoparticles production with ascorbic acid

![icon of a lightbulb and gearwheel](https://static.igem.wiki/teams/4259/wiki/engineering-page/motivation.svg)

Motivation

Binanox is a biomanufacturing system with many variables, which impels preliminary simplification to learn more about the process.

![icon of a lightbulb and gearwheel](https://static.igem.wiki/teams/4259/wiki/engineering-page/measure.svg)

Design

Investigate a more simple synthesis method before proceeding to a complex, bioreducing system.

![icon of a lightbulb and gearwheel](https://static.igem.wiki/teams/4259/wiki/engineering-page/wrench.svg)

Build & Test

Use Box-Behnken Design for modeling nanoparticle synthesis with ascorbic acid.

![icon of a lightbulb and gearwheel](https://static.igem.wiki/teams/4259/wiki/engineering-page/cap.svg)

Learn

Optimal gold:silver salts ratio for synthesis of PTT-optimal nanoparticles in biologically-relevant conditions is 1.85:1.

## Design

First, we decided to identify the optimal conditions for chemical nanoparticle synthesis using ascorbic acid. This way we could investigate several parameters in a more simple setting before moving on to a complex biological system. Additionally, to bridge the gap between chemical and biological synthesis, the reduction with ascorbic acid was conducted at pH 7 instead of pH 3, as done in the reference paper2. This was done under the presumption that optimal functionality of reducing enzymes present in *Escherichia coli* (*E.coli*) is achieved at neutral pH, as it corresponds to the pH of optimal growth in this bacterium6. As this methodology deviates from the original paper, the ascorbic acid experiments were repeated at biologically relevant pH to obtain an optimal gold:silver (HAuCl4:AgNO3) ratio for this pH range.

We used Box-Behnken Design to minimize the number of experiments necessary to identify the optimal HAuCl4:AgNO3 ratio for the synthesis of nanoparticles with a plasmon peak at 800 nm. For a more detailed description of Box-Behnken Design, you can refer to the [Modeling page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D). We could find the optimum for three variables in just 15 experiments instead of 27. The three variables were (1) the concentration of AgNO3 (2) the concentration of HAuCl4, and (3) pH. The latter was varied between 5 and 9 to test the influence of pH on the formation of nanoparticles under biologically relevantpH conditions6.

## Build & Test

After synthesis, the 450-1000 nm absorption spectrum was measured for each sample. These spectra were used as input for the model, which then returned a correlation between the three variables and the absorbance, displayed as an interactive slider in Fig. 3. This yielded a prediction of the spectrum, while the main optimization objective was a high absorbance at 800 nm. You can refer to the [Modeling page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D) for a detailed discussion of the methodology and the results of this experiment.

**pH:**

5

7

9

**Silver (mM):**

0.05

0.10

0.15

**Gold (mM):**

0.15

0.20

0.25

Maximize A800 nm

Maximize A800 nm at pH ~7.3

**Fig. 3** |
Results of the model for nanoparticle production using ascorbic acid, HAuCl4 and AgNO3, while varying the pH. The slider allows for adjusting the gold and silver salts concentrations and the pH, while dynamically viewing the influence of those conditions on the absorption spectrum of the synthesized nanoparticles.

If you enjoy tweaking this slider, check out our [Modeling page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D#slider2)! Here, you can check the effect of pH, temperature, and gold concentration on the absorbance of nanoparticles in a cell-free system. This tool can be useful for setting up a production process according to prerequisites. This is an important step in the upscaling of the biomanufacturing process.

## Learn

The model predicted an optimal molar ratio of HAuCl4:AgNO3 = 1.80:1 at a pH of 6.1. However, as will be elaborated in Cycle 2, Mueller Hinton (MH) Broth was shown to be an optimal growth medium for nanoparticle production. The pH of MH Broth is 7.3, thus the model was adjusted to maximize A800 nm for this pH value. This yielded a ratio of 1.85:1. Subsequently, the spiky morphology of the prepared nanoparticles was confirmed using TEM (Fig. 4). This “golden ratio” was used for all subsequent experiments with the biological system, because the produced nanoparticles in this experiment had the correct shape and size to observe plasmon resonance at 800 nm.

![](https://static.igem.wiki/teams/4259/wiki/engineering-page/chem-tem.jpg)

**Fig. 4** | A TEM micrograph of a spiky nanoparticle synthesized with ascorbic acid and ratio of gold:silver ions of 1.85:1.

# Cycle 2. Getting the right biological system for nanoparticle production

![icon of a lightbulb and gearwheel](https://static.igem.wiki/teams/4259/wiki/engineering-page/motivation.svg)

Motivation

A literature protocol for short nanoparticle synthesis with MH broth did not work 7.

![icon of a lightbulb and gearwheel](https://static.igem.wiki/teams/4259/wiki/engineering-page/measure.svg)

Design

Adjust the nanoparticle synthesis protocol to span over 24h and test 4 common growth media.

![icon of a lightbulb and gearwheel](https://static.igem.wiki/teams/4259/wiki/engineering-page/wrench.svg)

Build & Test

Test nanoparticle synthesis with lysed and non-lysed supernatants of wildtype *E. coli* grown in 4 broths: LB, LB low salt, MH, TSBS.

![icon of a lightbulb and gearwheel](https://static.igem.wiki/teams/4259/wiki/engineering-page/cap.svg)

Learn

Non-lysed MH broth yielded the best performance in PTT-optimized nanoparticle synthesis.

## Design

The next step in developing the biomanufacturing system was establishing which growth medium allows the model organism *E. coli* BL21 to produce the most effective blend of reducing agents. Extensive literature reviews show that the reducing agents play a crucial role in the size and morphology of the produced nanoparticles 4, which demands fine-tuning the bioreducing system.

Gurunathan et al.8 has shown the capacity of *E. coli* supernatant to produce silver nanoparticles from AgNO3 within five minutes7. The supernatant was obtained after 24h of growth in MH broth. After unsuccessfully attempting this conveniently short synthesis method, another paper stating that nanoparticle synthesis occurred in 24h was consulted8. Preliminary tests showed that this timespan was indeed sufficient. Therefore, after this point, all biochemical synthesis was performed over 24h.

To assess the optimal media for bacterial growth, we performed nanoparticle synthesis with supernatants of four different media: LB, LB Low Salt, MH, TSBS. Another variable tested was whether it is better to lyse the cells used for nanoparticle production. Lysing of the cells can be achieved by sonicating the cell liquid culture before separating the supernatant from the pellet. The [Results page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D#exp-6) further discusses the choice of media and sonication settings.

## Build & Test

The optimal medium for nanoparticle production was chosen based on the best absorbance at 800 nm and further confirmed with Transmission Electron Microscopy (TEM). An overview of the main findings is presented below, see the bar plots with A800 nm of different media (Fig. 5) and TEM micrographs (Fig. 6). A complete description of our results can be found on the [Results page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D#exp-4).

[![](https://static.igem.wiki/teams/4259/wiki/engineering-page/fig4-1.png)](https://static.igem.wiki/teams/4259/wiki/engineering-page/fig4-1.png)

**Fig. 5** | Bar plots showing the absorbance at 800 nm for samples of nanoparticles synthesized with supernatants of wild type *Escherichia coli* BL21 grown inLB, LB Low Salt, MH and TSBS media. A comparison was made between the addition and exclusion of cell lysate in the supernatant. Error bars represent standard error (n = 3). See Results pages for full spectra, including those of control samples.

[![](https://static.igem.wiki/teams/4259/wiki/engineering-page/media-tem.jpg)](https://static.igem.wiki/teams/4259/wiki/engineering-page/media-tem.jpg)

**Fig. 6** | An overview of TEM micrographs of nanoparticles produced with the non-lysed supernatant of MH Broth and LB. An overview and a zoom-in on one particle are provided.
To see all recorded TEM pictures for this experiment, see Notebook.

## Learn

MH supernatant without the lysate was found to have the best capacity for producing nanoparticles with the desired morphology (see Fig. 6 for spiky spheres) and a plasmon peak at 800 nm. Therefore, this engineering cycle helped us establish another parameter for Binanox: the optimal medium for bacterial growth of nanoparticle-producing *E. coli*.

It was also noticed that the control samples with media, silver, and gold salts could also produce nanoparticles. Therefore, to test which constituents of the media were responsible for the reducing capacity, another experiment was set up, as described on the [Results page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D#exp-9).

# Cycle 3. Boost Binanox with synthetic biology

![icon of a lightbulb and gearwheel](https://static.igem.wiki/teams/4259/wiki/engineering-page/motivation.svg)

Motivation

Nanoparticle synthesis can be further improved by overexpression of proteins that contribute to bioreduction of metal salts.

![icon of a lightbulb and gearwheel](https://static.igem.wiki/teams/4259/wiki/engineering-page/measure.svg)

Design

Adjust the nanoparticle synthesis protocol to span over 24h and test 4 common growth media.

![icon of a lightbulb and gearwheel](https://static.igem.wiki/teams/4259/wiki/engineering-page/wrench.svg)

Build & Test

Test the effect of adding a pellet with overexpressed proteins to the bioreducing mix on nanoparticle synthesis.

![icon of a lightbulb and gearwheel](https://static.igem.wiki/teams/4259/wiki/engineering-page/cap.svg)

Learn

Adding the cell lysate to the bioreduction mix lessens its reducing capacity. Consider ensuring secretion of the overexpressed proteins.

After establishing the optimal medium and treatment of the biological system for nanoparticle production, we proceeded to the next step: further improving the biomanufacturing of nanoparticles by adding specific metal-reducing proteins. As seen in the literature, *Cupriavidus metallidurans* proteins CopA 9, 10 and NapA 11, 12, 13 have been studied in nanoparticle synthesis. In addition, the *cup1* gene from *Candida albicans* was also shown to improve nanoparticle synthesis14. The beneficial effect of these proteins is mainly achieved through the increase in the reduction capacity of the mix. This typically leads to a higher yield and can influence the morphology4.

To test if the overexpression of these genes has a beneficial effect on nanoparticle synthesis in Binanox, we designed plasmids with the genes of *C. albicans* and *C. metallidurans*. Unfortunately, the transformations could not be performed within the limited lab time. You can read more about this on the [Results page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D#exp-6). This led us to substitute these genes with analogs from *E. coli* obtained from the ASKA library15. This way, we tested *copA*, *napA*, *cueO* and, additionally, *melA*, which was also found to have the potential to improve nanoparticle synthesis16.

**ASKA Library:** **A** complete **S**et of E. coli **K**-12 ORF **A**rchive. This library contains E. coli K-12 clones with every individual gene of E. coli, fused with a His-tag and, optionally with GFP. The genes are also framed by an IPTG-inducible promoter and Sfil restriction sites. At Binanox, we utilized this library for overexpression of E. coli proteins CopA, NapA, CueO and MelA.

## Design

To test the effect of the selected genes on nanoparticle production, we isolated the ASKA pCA24N plasmids from the library-standard *E. coli* K12 and cloned them into our *E. coli* BL21 strain. This allowed us to overexpress these genes. The proteins were all expressed intracellularly, so after IPTG induction, the pellet of a liquid culture would contain a high concentration of the protein of interest. After this, the pellet was resuspended in a smaller volume and lysed through sonication. Finally, this pellet lysate containing the overexpressed protein of interest was added to the MH supernatant of *E. coli* BL21 with an empty pET16b plasmid (introduced for antibiotic resistance). We then added silver and gold salts to this reaction mix to study nanoparticle formation.

The expected effect of all proteins on nanoparticle synthesis was a higher yield due to an increase in the reduction capacity of the mix. However, a change in the reaction mix can lead to changes in the synthesized nanoparticles' morphology, size distribution, and composition4. In this cycle, we directed our attention to the yield of nanoparticles with an absorbance at 800 nm, expressed in the intensity of A800 nm. Such an inference could be made after a supplementary experiment that found a direct correlation between nanoparticle concentration and absorbance (for more information, see the [Results page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D#exp-2)).

## Build & Test

Upon confirmation of the IPTG-induced overexpression of the proteins (see [Results page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D#fig-27) for SDS gel), nanoparticle synthesis was performed according to the methodology described above. Additionally, when MelA was used for synthesis, L-DOPA was added, as MelA converts L-DOPA into the reducing agent melanin. Finally, absorption spectra were measured and compared to the controls without adding the proteins (Fig. 7).

[![](https://static.igem.wiki/teams/4259/wiki/engineering-page/fig6-1.png)](https://static.igem.wiki/teams/4259/wiki/engineering-page/fig6-1.png)

**Fig. 7** | 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 page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D#exp-6) for full spectra, including those of control samples.

## Learn

All the tested ASKA genes showed a significant decrease in A800 nm when added to MH (p < 0.05). By contrast, when added to WT *E. coli* BL21 supernatant, no significant difference (p > 0.20) was observed compared to the control. Moreover, most samples leaned towards a decrease in A800 nm, except for MelA with L-DOPA. MelA-containing lysate resulted in a significant increase for A800 nm compared to its control (p < 0.0005), which contained a lysate of WT. However, this increase was insignificant compared to the control without any lysate (p = 0.317). Observing that the absorbance becomes lower upon the addition of lysate is peculiar.

A potential clarification for this decline in nanoparticle producing capacity is the interaction of the overexpressed proteins with other compounds in the reducing mix, which causes the overall mix to be less effective in synthesizing nanoparticles. Such compounds could be other oxidizing agents that compete with metal ions. Alternatively, proteases could be disturbing the functionality of the reducing peptides. Both scenarios lead to a decrease in nanoparticle yield, as the reduction of metal ions experiences a significant drop. A more elaborate discussion of this prospect is discussed on the [Results page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D#exp-6). In addition, the integration of these results into Binanox is discussed in Cycle 4.

# Cycle 4. Further improving the Binanox system with nitrate-containing media

![icon of a lightbulb and gearwheel](https://static.igem.wiki/teams/4259/wiki/engineering-page/motivation.svg)

Motivation

Nitrate-containing media induces the production of metal-reducing enzymes in *E. coli*.

![icon of a lightbulb and gearwheel](https://static.igem.wiki/teams/4259/wiki/engineering-page/measure.svg)

Design

Test the influence of *E. coli* supernatant grown on MH-Nitrate on nanoparticle synthesis.

![icon of a lightbulb and gearwheel](https://static.igem.wiki/teams/4259/wiki/engineering-page/wrench.svg)

Build & Test

Compare the effect of supernatant of *E. coli* grown on MH vs MH-Nitrate on nanoparticle synthesis.

![icon of a lightbulb and gearwheel](https://static.igem.wiki/teams/4259/wiki/engineering-page/cap.svg)

Learn

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

The last step we undertook to optimize our biomanufacturing process was ensuring a better reducing power of the supernatant, to which we added the lysate with overexpressed proteins. According to a paper by Gurunathan et al.8, the presence of nitrate in the medium during growth increases the reducing capacity of the supernatant due to an increase in nitrate reductase production by the organism8. In addition, nitrate reductases, such as NapA, are responsible for silver nanoparticle synthesis17. Following this train of thought, we decided to test whether adding nitrate to MH would improve the yield of nanoparticle production by the supernatant-lysate mix.

## Design

We compared the performance of the total ASKA-protein-containing bioreduction mix with the supernatant of MH to the supernatant of MH-Nitrate. We hoped to see an increase in the A800 nm of the obtained samples, which would signify that more nanoparticles with the desired properties are being synthesized.

## Build & Test

400-1000 nm absorption spectra were measured for all reducing blends. The addition of the supernatant of MH was compared to the addition of the supernatant of MH-Nitrate. Fig. 8 shows the A800 nm for all samples that contained gold and silver salts.

[![](https://static.igem.wiki/teams/4259/wiki/engineering-page/fig7-1.png)](https://static.igem.wiki/teams/4259/wiki/engineering-page/fig7-1.png)

**Fig. 8** | Bar plots showing the absorbance at 800 nm for samples of nanoparticles synthesized with supernatant of MH or MH-Nitrate, with addition of overexpressed proteins: CopA, CueO, napA and MelA. A comparison was held between samples that contain a supernatant of a liquid culture grown on MH or MH-Nitrate. Error bars represent standard error (n = 3). See [Results page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D#exp-7) for full spectra, including those of control samples.

## Learn

None of the samples showed a significant change in the absorbance at 800 nm between MH and MH-Nitrate supernatant (p > 0.05). This was an unexpected result, but we see potential explanations. One possibility is that nitrate's effect on these systems was insignificant due to the overriding reducing capacity of the overexpressed ASKA genes, which deems the influence of other reducing agents negligible. Another possibility is that similarly to the experiment in Cycle 3, the presence of the cell lysate in the mix introduced other oxidizing agents into the sample, which decreased the metal-reducing capacity of supernatant grown on MH.

The main conclusion from Cycles 3 and 4 is the necessity to eliminate the probable detrimental effects of the lysate on the reducing blend. Therefore, an alternative way to add the overexpressed proteins to the mix is necessary, for example, by tagging the proteins with secretion tags. This way, they would make up a part of the supernatant, which is expected to show an even better performance in reducing nanoparticles.

# Conclusion

Going through the four engineering cycles, we got to learn more and more about the optimization of our nanoparticle biomanufacturing system Binanox. This invaluable information for process design could not have been obtained without getting negative results from the setup experiments, learning from them, and adjusting the system accordingly. Although each cycle helped us obtain important results individually, the first two cycles were used as input for the setup of the following two cycles. Moreover, all of the conducted experiments led us to conduct experiments to further investigate the matter of enhancing the synthesis of PTT-optimized nanoparticles.

# Future experiments

## Introduction

As an iGEM project only has a duration of a few months, there is not enough time
to fully produce a complex product like a manufacturing process or a therapy.
Hence, our team has looked further than just the end of iGEM and proposed future
experiments. These future experiments are inspired by conversations with
[stakeholders](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D) and show what steps need to be taken to establish a final
product. We have identified three areas of research that will require further
study.

## Biomanufacturing

**Goal:** Improve upon our current production process.

**Design - Cell-free:** During our lab time we did not use a
protease inhibitor to prevent the degradation of our reducing proteins when
lysing the bacterial cells. This likely caused the unfortunate results in our
[cell-free experiments](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D#exp-4). Therefore, we would
like to do more experiments to further explore the cell-free production using a
protease inhibitor.

**Design - Simultaneous reduction:** To improve upon our
production design, we could in the future use ion-specific enzymes. With the
current method of our production process and that of chemical synthesis, gold is
reduced faster than silver. To enable simultaneous reduction of gold and silver
the use of ion specific enzymes could be the key18.

**Design - Upscaling:** We should adapt our production process in
order to obtain a consistent nanoparticle yield over different batches. This
includes doing upscaling experiments, whereby we will aim to obtain stable
concentrations in 10 L volumes. These results are then scaleable to 1000 L
[bioreactors](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27implementation%27%29%20%7D%7D#sustainability).

**Design - Isolation and Sterilization:** Furthermore, we need to
find an optimal isolation and sterilization method to obtain clean nanoparticles
that can be utilized in medical applications. To obtain isolated nanoparticles
we could use an ultracentrifuge or nanocentrifuge dependent on the weight of our
nanoparticles. In regard to obtaining sterilized nanoparticles, potential
methods are UV light and filter sterilization. Some of these methods have been
tested. However, an expansion on these experiments would be vital to the success
of our nanoparticles.

## Analysis

**Goal:** Further characterize nanoparticles.

**Design:** Nanoparticle behavior is highly dependent on their
size, shape and surface area19. Therefore, there are some additional
measurements that we could do to get a better understanding of our
nanoparticles. As can be read in Human Practices, we spoke to an expert about
the NTA, DLS and Nanocet-1. Each of these measures can determine nanoparticle
size. Furthermore, if we decide to explore the opportunity of a diagnostic
application further, we should determine the density of our nanoparticles. We
could use a method like Small Angle X-ray Scattering (SAXS)20.
Lastly, literature indicated the collapsing of spikes due to heat. It is
interesting to discover at which temperature and duration of heating this will
occur and whether it will influence our implementation.

## Toxicity

**Goal:** To accurately model *in vivo* responses to our
nanoparticles.

**Design:** To make sure our nanoparticles are safe for *in vivo*use, we need to determine whether coating has a significant effect on
toxicity. To further elucidate toxicity and photothermal therapy effectiveness
*in vitro* experiments using both cancer and non-cancer cell lines are
desired. We will have to test different concentrations of nanoparticles and the
duration of heating during the *in vitro* experiments. Additionally, to
comply with EMA regulations we will have to investigate cyto- and genotoxicity.
During *in vivo* experiments, we will also have to investigate the
ability of our nanoparticles to infiltrate tumor tissue and the effects of
antibody-nanoparticle conjugation on surface plasmon resonance and the
effectiveness of PTT.

# References

1. Sudha, P. N., Sangeetha, K., Vijayalakshmi, K., & Barhoum, A. Nanomaterials history, classification, unique properties, production and market. *Emerging Applications of Nanoparticles and Architecture Nanostructures*, 341–384 (2018)- 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).- Yeh, Y. C., Creran, B., & Rotello, V. M. Gold nanoparticles: preparation, properties, and applications in bionanotechnology. *Nanoscale* **4**, 1871–1880 (2012). - Rodrigues, T. S. *et al.* Synthesis of Colloidal Metal Nanocrystals: A Comprehensive Review on the Reductants. *Chemistry – a European Journal*, **24**, 16944–16963 (2018). - Zhang, D. Green Synthesis of Metallic Nanoparticles and Their Potential Applications to Treat Cancer. *Frontiers* **8** (2020).- Davey, K. Modelling the combined effect of temperature and pH on the rate coefficient for bacterial growth. *International Journal of Food Microbiology* **23**, 295–303 (1994).- Shahverdi, A. R., Minaeian, S., Shahverdi, H. R., Jamalifar, H., & Nohi, A. A. Rapid synthesis of silver nanoparticles using culture supernatants of Enterobacteria: A novel biological approach. *Process Biochemistry* **42**, 919–923 (2007).- Gurunathan, S. *et al.* Biosynthesis, purification and characterization of silver nanoparticles using Escherichia coli. *Colloids and Surfaces B: Biointerfaces* **74**, 328–335 (2009).- Bütof, L. *et al.* Synergistic gold–copper detoxification at the core of gold biomineralisation in Cupriavidus metallidurans. *Metallomics*, **10**, 278–286 (2018). - Reith, F. *et al.* Mechanisms of gold biomineralization in the bacterium Cupriavidus metallidurans. *Proceedings of the National Academy of Sciences* **106**, 17757–17762 (2009).- Vaidyanathan, R. *et al.* Enhanced silver nanoparticle synthesis by optimization of nitrate reductase activity. *Colloids and Surfaces B: Biointerfaces* **75**, 335–341 (2010).- Khodashenas, B. Nitrate reductase enzyme in Escherichia coli and its relationship with the synthesis of silver nanoparticles. *Journal of Research in Science, Engineering and Technology* **3**, 26–32 (2019).- Fu, J. K., Zhang, W. D., Liu, Z. Y., Yao, B. X., & Weng, S. Z. Characterization of adsorption and reduction of noble metal ions by bacteria. *Chin J Chem Univ* **20**, 1452–1454 (1999).- Yuan, Q. Enhanced Silver Nanoparticle Synthesis by Escherichia Coli Transformed with Candida Albicans Metallothionein Gene. *Material* **12**, 4180 (2019).- Kitagawa, M. *et al.* Complete set of ORF clones of Escherichia coli ASKA library (A Complete Set of E. coli K-12 ORF Archive): Unique Resources for Biological Research. *DNA Research* **12**, 291–299 (2006).- Tsai, Y. J. *et al.* Biosynthesis and display of diverse metal nanoparticles by recombinant Escherichia coli. *RSC Adv.* **4**, 58717–58719 (2014).- Anil Kumar, S. *et al.* Nitrate reductase-mediated synthesis of silver nanoparticles from AgNO3. *Biotechnology Letters* **29**, 439–445 (2007).
                                 - Esmail, R., Afshar, A., Morteza, M., Abolfazl, A. & Akhondi, E.
                                   Synthesis of silver nanoparticles with high efficiency and stability by culture
                                   supernatant of Bacillus ROM6 isolated from Zarshouran gold mine and evaluating
                                   its antibacterial effects. *BMC Microbiol* **22**, (2022).
                                 - Jeevanandam, J., Barhoum, A., Chan, Y. S., Dufresne, A. & Danquah, M. K.
                                   Review on nanoparticles and nanostructured materials: history, sources, toxicity
                                   and regulations. *Beilstein journal of nanotechnology*
                                   **9**, 1050–1074 (2018).
                                 - Minelli, C. et al. Measuring the size and density of nanoparticles by
                                   centrifugal sedimentation and flotation. *Analytical methods*
                                   **10**, 1725–1732 (2018).

{% endblock %}


{% extends "layout.html" %} {% block title %}Results{% endblock %} {% block
page\_content %}

# {{ self.title() }}

An overview of our experimental workflow

## Optimization

Finding suitable reaction conditions for biological production of nanoparticles

## Experiments

Testing our engineered microbial factory

## Upscaling

Aiming for industrial scale production

* CONTENTS
* [Introduction](#introduction)
* [Chemical nanoparticles](#character)
* [Experiments with WT
  *E. coli* BL21](#establishing)
* [Experiments with
  *E. coli* genes](#idc)
* [Supplementary](#supp)
* [Upscale & Extraction](#impl)
* [Conclusion](#concluck)
* [References](#ref)

# Introduction

With Binanox, we aimed to establish a
**biomanufacturing system**
for the production of metallic nanoparticles optimized for
photothermal therapy (PTT). Upon conducting a thorough literature
review, we established an experimental foundation for our project.
On this page, we describe the most important results that portray
our journey while designing Binanox.

![ask moor](https://static.igem.wiki/teams/4259/wiki/results/fig1.png)

**Fig. 1 |** Visual representation experimental
flow of the project.

# Characterization of chemically synthesized nanoparticles

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

![icon of a lightbulb and gearwheel](https://static.igem.wiki/teams/4259/wiki/results/goal.png)

Goal

Establish optimal conditions for PTT-optimized nanoparticles
production using ascorbic acid.

![icon of a lightbulb and gearwheel](https://static.igem.wiki/teams/4259/wiki/engineering-page/wrench.svg)

Method

Use Box-Behnken Design for modeling nanoparticle synthesis
with ascorbic acid.

![icon of a lightbulb and gearwheel](https://static.igem.wiki/teams/4259/wiki/results/hypothesis.png)

Hypothesis

Molar ratio of 10:1 is optimal at pH 3, according to
literature3.

![icon of a lightbulb and gearwheel](https://static.igem.wiki/teams/4259/wiki/engineering-page/cap.svg)

Conclusion

Molar ratio of 1.85:1 yields the highest A800 nm.

Previous research findings revealed that 800 nm is the optimal
absorbance wavelength for nanoparticles for photothermal
therapy1. To find the synthesis conditions that
**optimize the absorbance at 800 nm** we applied a
statistical model called Box-Behnken Design (BBD). The details on
the theoretical background can be found on the
**[Modeling page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D).** According to literature, pH, temperature, and gold and silver
salts concentration have been shown to have a significant
influence on nanoparticle formation2,3,4. Therefore,
these conditions were chosen to be varied.

Experiments were performed in two iterations
[(find out more on the modeling page)](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D). In the first iteration, where we tested ascorbic acid
nanoparticle synthesis, gold and silver salts concentration and pH
were varied. Identifying the optimum of these parameters for a
simple chemical system allowed us to obtain a solid basis before
moving on to more complex experiments with a bioreducing system.
Thus, the model was used to find the
**optimal HAuCl4:AgNO3 ratio at the pH of
the MH broth**, which was identified to be optimal for nanoparticle production.

Prior to starting the modeling experiment, a preliminary
experiment was performed in order to establish a suitable
HAuCl4:AgNO3. Ascorbic acid was added to
varying concentrations of HAuCl4 and AgNO3,
varying concentrations were obtained by diluting these salts in
ddH2O. Fig. 2 shows how this affected absorbance at 800
nm. The
**highest average absorbance was measured with 0.20 mM HAuCl4
and 0.10 mM AgNO3**; therefore, these values were chosen as the central values for
Box-Behnken Design. At the same time, the absorbance was high at
0.05 mM and 0.15 mM AgNO3. Therefore, we decided to
make a range of 0.20±0.05 mM for HAuCl4 and 0.10±0.05
mM for AgNO3.

![Graph with absorbance measurements of               nanoparticles produced using ascorbic acid.](https://static.igem.wiki/teams/4259/wiki/results/fig2.png)

**Fig. 2 |** Absorbance measurements of
nanoparticles produced using ascorbic acid with six
different gold and silver salts concentrations (n = 3). The
average absorbance at 800 nm with the standard error of the
mean was plotted. The highest average absorbance at 800 nm
was found for a ratio of 0.20 mM gold and 0.10 mM silver.

Upon obtaining the concentration range, the modeling experiment
was started. The absorption spectra of this experiment can be
found in Fig. 3, using the sliders, the effect of different
combinations of conditions on the absorption spectra is shown.
It is visible that pH has a drastic impact on the absorption
spectrum. In the Design Expert software, A800 nm was
optimized with one other criterion; the pH should be 7.3±0.05
since it is the pH of MH broth. This gave the optimal parameter
values: **pH 7.25**, [HAuCl4] = 0.19 mM,
and [AgNO3]= 0.10 mM. This way, the
obtained ratio **HAuCl4:AgNO3 ≈ 1.85:1**. This
ratio was used in all subsequent experiments.

**pH:**

5

7

9

**Silver (mM):**

0.05

0.10

0.15

**Gold (mM):**

0.15

0.20

0.25

Maximize A800 nm

Maximize A800 nm at pH ~7.3

**Fig. 3** |
Absorption spectrum of chemically synthesized nanoparticles for different pH, [HAuCl4], and [AgNO3] using ascorbic acid.

## 2. Obtaining a correlation between nanoparticle concentration and absorbance for nanoparticles synthesized with ascorbic acid

![icon of a lightbulb and gearwheel](https://static.igem.wiki/teams/4259/wiki/results/goal.png)

Goal

Show that a higher absorbance value corresponds with a
higher concentration of nanoparticles.

![icon of a lightbulb and gearwheel](https://static.igem.wiki/teams/4259/wiki/engineering-page/wrench.svg)

Method

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

![icon of a lightbulb and gearwheel](https://static.igem.wiki/teams/4259/wiki/results/hypothesis.png)

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.

![icon of a lightbulb and gearwheel](https://static.igem.wiki/teams/4259/wiki/engineering-page/cap.svg)

Conclusion

A correlation between absorbance and nanoparticle
concentration was observed, with A = A0\*(½)n
and an R2-value of 0.93.

In order to clearly interpret the absorption spectra of our
nanoparticles, we performed a serial dilution to see if it
correlated with the absorbance. This would essentially tell us
if a higher absorbance also means a higher nanoparticle
concentration. Therefore, AgNO3 and HAuCl4 were dissolved in ddH2O, by using the molar ratio of
gold and silver 1.85:1.

The absorption spectrum was measured for each dilution sample.
Fig. 4A shows the absorbance against wavelength. The results
showed, for all the dilutions, a clear peak at 400 nm, in which
a higher dilution resulted in a lower absorbance. Fig. 4B shows
a correlation of the format A = A0\*(½)n with an
R2-value of 0.93. This leads to the conclusion that
there is a correlation between the absorbance and the
concentration of nanoparticles. Meaning, the more nanoparticles
present in solution the higher the absorbance.

![Graph showing absorbance against wavelength of                 different diluted samples and graph with linear correlation](https://static.igem.wiki/teams/4259/wiki/results/fig4.png)

**Fig. 4 |** A.) Graph showing absorbance
against wavelength of different diluted samples of
chemically produced nanoparticles using ascorbic acid with a
gold:silver ratio of 1.85:1. A peak at around 478 nm is
observed for every sample. A higher dilution indicates a
lower absorption. B.) Per dilution the absorbance at 478 nm
was plotted and the R2 calculated, showing a linear
correlation.

## 3. Recording dynamic absorbance measurements of nanoparticle synthesis using ascorbic acid

![icon of a lightbulb and gearwheel](https://static.igem.wiki/teams/4259/wiki/results/goal.png)

Goal

Record the shifting of the peak, within the absorbance
against the wavelength graph, to the left with time for
nanoparticle synthesis using ascorbic acid.

![icon of a lightbulb and gearwheel](https://static.igem.wiki/teams/4259/wiki/engineering-page/wrench.svg)

Method

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

![icon of a lightbulb and gearwheel](https://static.igem.wiki/teams/4259/wiki/results/hypothesis.png)

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.

![icon of a lightbulb and gearwheel](https://static.igem.wiki/teams/4259/wiki/engineering-page/cap.svg)

Conclusion

The peak shifted towards 625 nm as the time progressed.
The shifting stabilized after approximately one hour.

After repeatedly getting peaks at ~600 nm when using the
bioreduction system, we questioned whether time had an influence
on the position of the peak. For this, we conducted synthesis of
nanoparticles with ascorbic acid over a span of 24 hours, while
measuring the absorption spectrum every 2-3 minutes (Fig. 5).
The data is only presented for the first hour, as the spectrum
did not change after the initial 60 minutes.

This data shows that, even though the synthesized nanoparticles
initially have a shape and size that results in a plasmon peak
at ~700 nm, it shifts towards 600 nm and remains stable there.
This can be clarified by a
**low thermal stability** of nanoparticles with a
branched morphology. Nanoparticles with a branched morphology
have a high surface energy, which leads to them readily
collapsing even at room temperature5. This emphasizes
the demand for finding a fitting capping material, which would
prevent the nanoparticles from collapsing and
aggregating6. This way, nanoparticles will maintain
an absorbance in the desired 700-800 nm range and ensure the
highest light-to-heat conversion capacity due to the presence of
the spikes.

An example of such a capping material is chitosan6.
Besides providing stability to the synthesized nanoparticles, it
is biocompatible. Biocompatibility is an important feature in
medical applications. More information can be found on the
[Human Practices page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D#person15).

![Graph with dynamic absorbance measurements of nanoparticle                 synthesis with ascorbic acid.](https://static.igem.wiki/teams/4259/wiki/results/fig5.png)

**Fig. 5 |** Dynamic absorbance measurements of
nanoparticle synthesis with ascorbic acid over a span of 1h
at 25°C. Used conditions: [HAuCl4] = 2mM,
[AgNO3] = 1.08mM, [Asc. Acid] = 0.4mM. pH = 7.

# Experiments with wild type *E. coli* BL21

## 4. Establishing a methodology for obtaining wild type *E. coli* BL21 supernatant for nanoparticle production

![icon of a lightbulb and gearwheel](https://static.igem.wiki/teams/4259/wiki/results/goal.png)

Goal

Determine the optimal growth medium and the effect of adding
cell lysate for obtaining a bioreduction system capable of
synthesizing PTT-optimized nanoparticles.

![icon of a lightbulb and gearwheel](https://static.igem.wiki/teams/4259/wiki/engineering-page/wrench.svg)

Method

Test nanoparticle synthesis with supernatants of WT
*E. coli* BL21 grown in four different growth media
and the effect of adding lysed cells.

![icon of a lightbulb and gearwheel](https://static.igem.wiki/teams/4259/wiki/results/hypothesis.png)

Hypothesis

Supernatant of MH is expected to perform best according to
literature7.

![icon of a lightbulb and gearwheel](https://static.igem.wiki/teams/4259/wiki/engineering-page/cap.svg)

Conclusion

Supernatant of MH Broth without lysing the cells performs
best.

The first step in establishing a bioreduction system for producing
PTT-optimized nanoparticles was to identify how the growth medium
could influence the production of nanoparticles. To investigate
this matter, we compared four media: LB, LB low salt, MH, and
TSBS. These experiments were done with the wild-type (WT) strain
*Escherichia coli* (*E. coli*) BL21, which was grown
in four different broths overnight at 37°C.

Supernatant was used to create a
**cell-free system** for nanoparticle production.
Additionally, it was tested whether including the cell lysate,
which may contain reducing agents, in the supernatant had a
beneficial effect on nanoparticle synthesis. A full description of
the experimental set-up can be found on the
[Notebook page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%20%7D%7D#core).

The absorption spectra were measured after 24h (Fig. 6). The
graphs indicate nanoparticle formation is
**less optimal when using lysed cells**. A possible
clarification is that the content of the cells may interfere with
nanoparticle formation or proteases released during lysis may have
denatured proteins capable of metal ion reduction. Furthermore,
**the supernatant of *E. coli* grown in MH broth showed a
higher absorbance**, indicating that this medium is beneficial for the production of
nanoparticles. Notably, the medium alone also resulted in high
absorbance, which suggests that the medium could also reduce the
silver and gold salts to some extent.

![Graphs with absorbance against wavelength for four different media.](https://static.igem.wiki/teams/4259/wiki/results/fig6.png)

**Fig. 6 |** Absorbance against wavelength for
*Escherichia coli* BL21 grown in four different media
containing silver and gold salts. The four different media
tested were: Mueller Hinton (MH) broth, Lysogeny broth (LB),
LB low salt (LBls), and TSBS. Additionally, the graph compares
using lysed cells against using supernatants of the cells.

After the absorbance measurements were taken, TEM was used to
study the formed nanoparticles. TEM pictures for all media can be
found on the Notebook page. For synthesis in LB and MH broth, TEM
micrographs showed the formation of nanoparticles of which some
contained spikes (Fig. 7). The TEM pictures show a zoomed-out
overview of the formed nanoparticles (Fig. 7), as well as isolated
urchin-like nanoparticles for both LB (Fig. 7A) and MH (Fig. 7B).
The sample of nanoparticles produced by LB supernatant (Fig. 7A)
showed a higher degree of aggregation. Moreover, the spikes seen
on these nanoparticles were not as prominent as in MH broth (Fig.
7B). Additionally, the size observed for the nanoparticles was
around **100 nm,** which is a desirable size for PTT.
**Therefore, based on the absorbance results and the TEM
pictures, we proceeded with MH broth in subsequent
experiments**.

![Transmission Electron Microscopy images.](https://static.igem.wiki/teams/4259/wiki/results/fig7.png)

**Fig. 7 |** Transmission Electron Microscopy
images of nanoparticles formed in two different media using
supernatant obtained from *E. coli* BL21 liquid
culture. A) Three TEM pictures formed in Lysogeny broth (LB)
medium. Aggregates are formed, but distinctive nanoparticles
can be identified. B) Three TEM pictures of nanoparticles
formed in Mueller Hinton (MH) medium. No aggregates are
observed, formation of urchin-like nanoparticles. The size of
the nanoparticles is around 100 nm.

## 5. Obtaining optimal conditions for nanoparticle synthesis with the bioreduction system Binanox

![icon of a lightbulb and gearwheel](https://static.igem.wiki/teams/4259/wiki/results/goal.png)

Goal

Identify the optimal synthesis conditions for producing
nanoparticles with a high A800 nm, using
supernatant and lysate of WT *E. coli* BL21 grown in
MH broth.

![icon of a lightbulb and gearwheel](https://static.igem.wiki/teams/4259/wiki/engineering-page/wrench.svg)

Method

Use three equidistant values for the three variable
conditions to identify the optimum combination with the help
of Box-Behnken design.

![icon of a lightbulb and gearwheel](https://static.igem.wiki/teams/4259/wiki/results/hypothesis.png)

Hypothesis

A low pH is optimal2. A higher temperature is
optimal8. Based on literature, gold concentration
should have a local optimum6.

![icon of a lightbulb and gearwheel](https://static.igem.wiki/teams/4259/wiki/engineering-page/cap.svg)

Conclusion

Maximization of A800 nm yielded pH 5.2,
[HAuCl4] = 5.75 mM, [AgNO3] = 3.11 mM
and T=49°C.

After obtaining the optimal medium for nanoparticle production
with the supernatant of WT *E. coli* BL21, we decided to
employ modeling to further
**optimize the Binanox system**. For this experiment,
we combined the knowledge obtained from iteration 1, described
above in [experiment 1](#exp-1), to
find the optimal pH, temperature, and metal salt concentration for
the production of nanoparticles with a high A800 nm.

Our previous experiments revealed the optimal gold:silver salts
concentrations ([experiment 1](#exp-1)), and that the non-lysed supernatant of WT *E.coli* BL21
grown on MH broth performed best as a bioreduction system ([experiment 4](#exp-4)). This obtained knowledge was used for the following experiment.
This experiment was performed using WT *E. coli* BL21
supernatant with *E. coli* BL21 lysate. The hypothesis was
that the overexpressed proteins would be present in higher
quantities after lysing the cells, increasing the reducing
potential. Before starting the experiment, it was necessary to
identify an adequate range for the optimizable parameters: pH,
temperature, and concentration of gold salts. More information
about the conditions and the ranges can be found below.

**pH**

Assuming that better protein function will result in better
nanoparticle formation, the optimal growth pH for *E. coli*
was used as the central value: pH 79. The adequate
range was further restricted by the biologically relevant pH
values for *E. coli*: growth is only possible between pH 5
and 97. Therefore, our tested pH values were 5, 7, and
9.

**Temperature**

Since 37°C is the optimal temperature for
*E. coli* growth it was chosen as the central value for the
temperature range10. In addition, literature showed
that nanoparticle formation benefited from higher
temperatures8. Therefore, the upper boundary was set to
49°C, which was the highest the shakers in our lab could achieve.
Finally, according to Box-Behnken Design, an equidistant point was
selected, which made the final temperatures tested: 25, 37 and
49°C.

**Gold concentration**

After obtaining the optimal gold:silver salts ratio in the first
iteration (1.85:1), it was necessary to establish which
concentration of gold and silver was optimal for PTT-optimized
nanoparticle production using *E.coli*. To identify an
adequate concentration range to be tested in the modeling, it was
necessary to conduct a preliminary experiment. The preliminarily
tested range for the concentration of golden salts was 0-8 mM. The
results showed that A800 nm decreased as the
concentration increased (Fig. 8). Therefore, it was decided to
test 2, 4, and 6 mM for modeling.

![Effect of HAuCl4 concentration on the absorption spectrum of biologically produced nanoparticles.](https://static.igem.wiki/teams/4259/wiki/results/fig8.png)

**Fig. 8 |** Effect of HAuCl4
concentration on the absorption spectrum of biologically
produced nanoparticles. Experiments were performed with
*E. coli* BL21 supernatant and varying concentrations
of HAuCl4. Also, AgNO3 was added in a
ratio of 1:1.85 to the HAuCl4 concentration. The
absorbance was measured in a plate-reader after 24 hours. All
gold concentrations, except for 0 mM, were done with three
biological replicates. The dotted lines represent the standard
error of the mean. A) Full absorption spectrum. B) Bar plot of
the absorbance at 800 nm.

The defined adequate ranges for pH, temperature, and
[HAuCl4] are summarized in Table 1. The absorption
spectra of the different combinations of conditions are
represented in Fig 9. To find the optimal conditions, the A800 nm
of the supernatant with lysate was maximized in the software
[Design Expert](https://www.statease.com/software/design-expert/)
by Stat Ease. The maximization yielded**pH 5.2, gold concentration 5.75 mM,** and **temperature 49** °C as optimal conditions.

**pH:**

5

7

9

**Temperature, °C**

25

37

49

**Gold (mM):**

2

4

6

Maximize A800 nm

Maximize A800 nm & Minimize T°C

Maximize A800 nm & Minimize Gold

**Fig. 9** |
Absorption spectra of biologically synthesized nanoparticles using *Escherichia coli* supernatant and lysate for different pH, [HAuCl4], and [AgNO3].

Similar results have been found in other studies. Divya et al.,
performed a study in which pH and temperature were independently
varied to optimize for AgNO3 nanoparticle synthesis
with *E. coli*2. pH 5, 7, and 9 were tested and
pH 5 was found to be optimal, which is in agreement with the
optimal pH of 5.2 that we found. For temperature, they tested 25°C
and 37°C, of which 37°C performed better. In our experiments too,
higher temperatures were found to be more optimal. Work by Gou et
al. has found similar findings8. They performed silver
nanoparticle synthesis using
*Lysinibacillus sphaericus* and temperature from 20°C to
90°C. Between 20°C and 70°C, the absorbance peak increased as the
temperature increased.

# Experiments with *E. coli* genes *napA, melA, cueO* and *copA*

## 6. Testing the effect of adding a cell lysate containing overexpressed NapA, MelA, CueO and CopA proteins on nanoparticle synthesis

![icon of a lightbulb and gearwheel](https://static.igem.wiki/teams/4259/wiki/results/goal.png)

Goal

Examine the influence of *E. coli* proteins CopA,
NapA, CueO, and MelA on nanoparticle formation.

![icon of a lightbulb and gearwheel](https://static.igem.wiki/teams/4259/wiki/engineering-page/wrench.svg)

Method

Add a pellet containing overexpressed proteins to WT
*E. coli* BL21 of MH broth and examine the effect on
nanoparticle synthesis.

![icon of a lightbulb and gearwheel](https://static.igem.wiki/teams/4259/wiki/results/hypothesis.png)

Hypothesis

All proteins are expected to increase the nanoparticle yield
and have a pronounced effect on the particle size
distribution due to their influence on the reduction rate.

![icon of a lightbulb and gearwheel](https://static.igem.wiki/teams/4259/wiki/engineering-page/cap.svg)

Conclusion

The data showed that the addition of lysates rich with
protein does not results in better or improved nanoparticle
formation.

Given that the efforts to transform the metal ion reducing genes
from *C. metallidurans* and *C. albicans* were
unsuccessful (See experiment 10 below, under supplementary
Experiments), biological production of bimetallic nanoparticles
was attempted using the ASKA collection, which is explained under
[experiment 8](#exp-8). The genes
selected from this collection were napA, melA, cueO and copA.

**NapA**

NapA is a nitrate reductase often released by
*E. coli* and is capable of forming nanoparticles
biologically. The process involves NapA converting nitrate to
nitrite, which leads to the oxidation of Ag+ to
Ag2+, which is easier reduced to its elemental form.
This results in the reduction of silver ions to silver
nanoparticles11.

**MelA**

MelA is a tyrosinase that is capable of converting L-DOPA to
melanin. Melanin contains functional groups such as quinone and
semiquinone, that work as effective reducing agents to convert
gold ions to gold12.

**CopA and CueO**

CopA and CueO are proteins associated with copper ion regulation
in *E.coli* cells. These proteins are often involved in the
conversion of copper ions to its elemental form. However, research
has shown that upon exposure to gold ions, CopA is over-expressed
in *C. metallidurans*. Thus, we attempted to check if the
protein functions to reduce silver and gold ions in *E.coli*6,13.

We approached this experiment by using the supernatant from
BL21-pET16b overnight liquid culture, which was then supplemented
with concentrated protein extract from the ASKA strains. An
overview of the experiment is briefly explained below; for a more
detailed overview, read our
[Notebook](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%20%7D%7D).

Liquid cultures for BL21-pET16b and all ASKA strains were cultured
overnight in MH broth to an approximate OD600 of
0.6-0.8. The liquid culture for BL21-pET16b was spun down to
obtain the supernatant, which was further sterilized using filter
sterilization. In order to induce the production of proteins in
the ASKA strain, liquid cultures were re-inoculated in fresh MH
broth to an OD600 of 0.4 and induced with IPTG. These
liquid cultures were centrifuged to pellet the bacterial cells,
which were then sonicated to break open the cells to release the
expressed proteins. Finally, the experiment was executed in a
24-well plate, where the supernatant was separately supplemented
with each protein extract, AgNO3 and HAuCl4
salts in duplo. These plates were left in a shaking incubator for
24 hours before the absorption measurements were taken.

The graph below (Fig. 10) shows absorbance obtained for ASKA CopA
at 800 nm. High absorbance was observed for the samples containing
medium with salts. This can be attributed to the presence of
tryptone in the medium which acts as a strong reducing agent. Read
more on the role of each media component under the experiment
section,
[testing media for nanoparticle synthesis](#exp-9). The highest absorbance was observed when silver and gold salts
were added to the WT supernatant. However, upon the addition of
lysate to supernatant with gold and silver salts, the absorbance
value dropped. An even lower absorbance was recorded for lysate
with silver and gold. These results were in contrary to the
hypothesis, where a higher protein concentration resulted in
increased nanoparticle synthesis.

![CopA absorbance graph obtained at 800 nm after addition of                 gold and silver ions to different media.](https://static.igem.wiki/teams/4259/wiki/results/fig10.png)

**Fig. 10 |** Absorbance graph obtained at 800 nm
after addition of gold and silver ions to Mueller Hinton (MH)
broth media, BL21-pET16b supernatant, MH broth with copA
lysate and a combination of copA lysate with BL21-pET16b
supernatant. These readings were taken at 24h after the
addition of gold and silver salts.

The graphs in Fig. 11 & 12 show A800 nm obtained for
ASKA CueO and NapA. The results obtained are quite similar to
absorbance observed for CopA, as explained above. Thus, it is
highly likely that cellular components released upon lysis
interfere with protein function or nanoparticle synthesis.

![CueO absorbance graph obtained at 800 nm after addition of                 gold and silver ions to different media.](https://static.igem.wiki/teams/4259/wiki/results/fig11.png)

**Fig. 11 |** Absorbance graph obtained at 800 nm
after addition of gold and silver ions to Mueller Hinton (MH)
broth media, BL21-pET16b supernatant, MH-broth with CueO
lysate and a combination of CueO lysate and BL21-pET16b
supernatant. These readings were taken at 24h after the
addition of gold and silver salts.

![NapA absorbance graph obtained at 800 nm after addition of                 gold and silver ions to different media.](https://static.igem.wiki/teams/4259/wiki/results/fig12.png)

**Fig. 12 |** Absorbance graph obtained at 800 nm
after addition of gold and silver ions to Mueller Hinton (MH)
broth media, BL21-pET16b supernatant, MH broth with NapA
lysate and a combination of NapA lysate and BL21-pET16b
supernatant. These readings were taken at 24h after the
addition of gold and silver salts.

The graph below (Fig. 13) shows absorbance obtained for ASKA MelA
at 800 nm. As discussed earlier, MelA uses L-DOPA for metal ion
reduction. Thus, a final concentration of 1 mM L-DOPA was used to
test the effect on nanoparticle production in the presence of
MelA.

Silver and gold salts were added to either MH broth, supernatant
WT, and lysate from WT. These experiments showed similar results
to the data discussed for CopA, CueO and NapA. However, the
addition of L-DOPA to WT supernatant containing MelA lysate and to
MH with either lysate from WT or ASKA MelA strains, causes an
increase in absorbance at A800 nm. The wells containing
L-DOPA developed an orange color within a few minutes of addition,
which may explain the higher absorbance values. This color change
may be due to the conversion of L-DOPA to melanin12.

![MelA and L-DOPA absorbance graphs obtained at 800 nm after addition of                 gold and silver ions.](https://static.igem.wiki/teams/4259/wiki/results/fig13.png)

**Fig. 13 |** Absorbance graph obtained at 800 nm
after addition of gold and silver ions and/or L-DOPA to MH
broth media, BL21-pET16b supernatant, MelA lysate and a
combination of MelA lysate, BL21-pET16b supernatant. These
readings were taken at 24h after the addition of gold and
silver salts, at a ratio of 1.85:1 M.

# Absorption spectra from 400 nm to 1000 nm for each reaction condition

![ASKA absorption spectra of Escherichia coli BL21 supernatant.](https://static.igem.wiki/teams/4259/wiki/results/fig14.png)

**Fig. 14 |** Absorption spectra of
*Escherichia coli* BL21 supernatant with the
addition of lysate ASKA of different genes in
combination with silver and gold salts. Subplots were
made for each ASKA gene and the controls. The two
controls are Mueller Hinton (MH) broth and supernatant
wildtype. The different ASKA genes were: CopA, CueO,
MelA, and NapA. Lysates of the ASKA genes were measured
in MH borth or in combination with supernatant WT. For
all the samples, gold and silver salts were added in the
ratio 1.85:1. All these readings were taken 24 hours
after the experiment.

![MelA and L-DOPA absorption spectra of Escherichia coli BL21 supernatant.](https://static.igem.wiki/teams/4259/wiki/results/fig15.png)

**Fig. 15 |** Absorption spectra of
*Escherichia coli* BL21 supernatant with the
addition of lysate MelA in combination with L-DOPA and
silver and gold salts. Subplots were made for the
addition of L-DOPA and the controls. The two control
supernatant wildtype and supernatant WT with the
addition of lysate MelA. The lysate of MelA was measured
in Mueller Hinton (MH) borth or in combination with
supernatant WT. For all the samples, gold and silver
salts were added in the ratio 1.85:1. All these readings
were taken 24 hours after the experiment.

Our experiments have indicated that the addition of cell lysates
containing metal reducing proteins did not lead to increased
production of nanoparticles as hypothesized. It is highly likely
that the cell lysate contains components which interfere with
nanoparticle synthesis or protein function such as proteases.
Thus, it may be ideal to obtain purified aliquots of the proteins
of interest for testing nanoparticle production or using a cell
system for biological production of nanoparticle synthesis.

## 7. Testing the effect of using supernatant of WT *E. coli* BL21 grown on a MH-Nitrate on nanoparticle synthesis

![icon of a lightbulb and gearwheel](https://static.igem.wiki/teams/4259/wiki/results/goal.png)

Goal

Improve the nanoparticle yield by using supernatant grown on
MH broth nitrate.

![icon of a lightbulb and gearwheel](https://static.igem.wiki/teams/4259/wiki/engineering-page/wrench.svg)

Method

Compare absorption spectra obtained after bioreduction with
supernatant grown in MH to MH broth nitrate + ASKA protein
pellet lysate.

![icon of a lightbulb and gearwheel](https://static.igem.wiki/teams/4259/wiki/results/hypothesis.png)

Hypothesis

*E. coli* secretes more reducing agents in MH broth
nitrate, resulting in a faster formation of nanoparticles
and thus higher yield in the same time span.

![icon of a lightbulb and gearwheel](https://static.igem.wiki/teams/4259/wiki/engineering-page/cap.svg)

Conclusion

Supernatant of *E. coli* grown in MH broth nitrate
does not result in a significantly higher nanoparticle yield
for any of the ASKA genes.

This experiment was inspired by S. Gurunathan et al. where they
used LB nitrate for the production of silver nanoparticles. These
experiments aimed to identify whether nitrate in the MH broth,
identified as an optimal medium through our experiments, has an
impact on the production of nanoparticles7.

An identical approach as detailed under
[ASKA- Effect of adding pellet lysate](#exp-6)
was used, however, BL21-pET16b was cultured overnight in MH broth
supplemented with nitrate salts. Read more about the experimental
design and protocol in our
[Notebook](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%20%7D%7D).

The graph shown in Fig. 16 compares the A800 nm for
samples containing CopA lysate for nanoparticle production in MH
broth and MH broth supplemented with nitrate (MH broth nitrate).
For samples that contain medium with silver and gold salts, the
absorbance remains consistent. However, the addition of salts to
the MH broth nitrate supernatant causes an increase in the
absorbance compared to MH broth supernatant. It is likely that the
nitrate in the medium promotes the formation of nanoparticles.
Unfortunately, the addition of lysates to MH broth nitrate results
in an even lower absorbance when compared to MH broth with
lysates. It is possible that these differences are due to
variations in execution of the experiments or that the nitrate in
the medium interacts with the components of the cell lysate and
causes further interference in nanoparticle production.

![arplots of absorbance obtained at 800 nm.](https://static.igem.wiki/teams/4259/wiki/results/fig16.png)

**Fig. 16 |** Barplots of absorbance obtained at
800 nm (A800 nm) for CopA samples in Mueller Hinton
(MH) broth and MH broth nitrate. Absorbance measurements were
performed to assess the influence of adding nitrate on the
A800 nm. For all the samples, gold and silver salts
were added in the ratio 1.85:1. Also, the effect of adding
lysate CopA to the supernatant wiltype on the A800 nm
was tested. For the control, the medium was used. The
absorbance was measured 24 hours after the start of the
experiment.

The graph shown in Fig. 17 is for A800 nm for samples
containing CueO lysate for nanoparticle production in MH broth and
MH broth nitrate. For samples that contain medium with silver and
gold salts, the absorbance remains consistent, whereas the
absorbance for the supernatant increases. Interestingly, the
absorbance increases upon addition of lysate to supernatant from
MH broth nitrate. This perhaps suggests that CueO works better in
MH-nitrate and results in increased production of nanoparticles as
evidenced by a higher absorbance.

![Barplots of absorbance obtained at 800 nm](https://static.igem.wiki/teams/4259/wiki/results/fig17.png)

**Fig. 17 |** Barplots of absorbance obtained at
800 nm (A800 nm) for CueO samples in Mueller Hinton
(MH) broth and MH broth nitrate. Absorbance measurements were
performed to assess the influence of adding nitrate on the
A800 nm. For all the samples, gold and silver salts
were added in the ratio 1.85:1. Also, the effect of adding
lysate CueO to the supernatant wiltype on the A800 nm
was tested. For the control, the medium was used. The
absorbance was measured 24 hours after the start of the
experiment.

For samples containing NapA, the medium with silver and gold salts
shows again a consistent absorbance, as indicated in Fig.18.
However, the addition of lysate to the supernatant obtained from
MH broth nitrate shows a slight drop in absorbance. This may
suggest that the production of NapA may not be largely affected by
changes in media conditions. However, take note that the error
bars are quite large for MH broth nitrate supernatant with lysate.

![Barplots of absorbance obtained at                800 nm.](https://static.igem.wiki/teams/4259/wiki/results/fig18.png)

**Fig. 18 |** Barplots of absorbance obtained at
800 nm (A800 nm) for NapA samples in Mueller Hinton
(MH) broth and MH broth nitrate. Absorbance measurements were
performed to assess the infleunce of adding nitrate on the
A800 nm. For all the samples, gold and silver salts
were added in the ratio 1.85:1. Also, the effect of adding
lysate NapA to the supernatant wiltype on the A800 nm
was tested. For the control, the medium was used. The
absorbance was measured 24 hours after the start of the
experiment.

For samples prepared with MelA and L-DOPA, the medium with silver
and gold salts shows a similar absorbance, as indicated in Fig. 19.
However, the supernatants that are supplemented with either WT
lysate or MelA containing lysates, salts and L-DOPA show
differences in absorbance. However, the error bars recorded for
these experiments are quite broad, thus we cannot clearly state
whether changes in medium impact the performance of MelA in
nanoparticle production.

![Barplots of absorbance obtained at 800 nm.](https://static.igem.wiki/teams/4259/wiki/results/fig19.png)

**Fig. 19 |** Barplots of absorbance obtained at
800 nm (A800 nm) for MelA samples in Mueller Hinton
(MH) broth and MH broth nitrate. Absorbance measurements were
performed to assess the influence of adding nitrate on the
A800 nm. For all the samples, gold and silver salts
were added in the ratio 1.85:1. Also, the difference between
the addition of lysate wildtype and lysate MelA was tested in
combination with L-DOPA. For the control, the medium was used.
The absorbance was measured 24 hours after the start of the
experiment.

# Absorption spectra from 400 nm to 1000 nm for each reaction condition

## **MelA**

The graph below (Fig. 20) shows the absorption spectrum from
400 nm to 1000 nm for experimental samples testing the role
of MelA. As can be seen, the highest absorbance is achieved
at 600 nm for MHB-nitrate supernatant with lysate, salts and
L-DOPA.This absorption peak is not ideal for photothermal
therapy, however, the absorbance was still high at 800 nm.

![Absorption spectra for MelA samples                       in Mueller Hinton (MH) broth and MH broth nitrate.](https://static.igem.wiki/teams/4259/wiki/results/fig20.png)

**Fig. 20 |** Absorption spectra for MelA
samples in Mueller Hinton (MH) broth and MH broth
nitrate. Absorbance measurements were performed to
assess the influence of adding nitrate on the absorbance
at 800 nm (A800 nm). For all the samples,
gold and silver salts were added in the ratio 1.85:1.
Also, the difference between the addition of lysate
wildtype (WT) and lysate MelA to the supernatant WT was
tested in combination with L-DOPA. For the control, the
medium was used. The absorbance was measured 24 hours
after the start of the experiment.

## **NapA**

Similar to the data discussed above, the highest absorbance
was recorded at 600 nm (Fig. 21). However, addition of
lysate to supernatants with salts only resulted in the
highest peak for MH broth medium and not for MH-nitrate.
These results can also be seen in the bar graph above for
NapA (Fig. 18).

![Absorption spectra for NapA samples in Mueller Hinton                       (MH) broth and MH broth nitrate.](https://static.igem.wiki/teams/4259/wiki/results/fig21.png)

**Fig. 21 |** Absorption spectra for NapA
samples in Mueller Hinton (MH) broth and MH broth
nitrate. Absorbance measurements were performed to
assess the influence of adding nitrate on the absorbance
at 800 nm (A800 nm). For all the samples,
gold and silver salts were added in the ratio 1.85:1.
Also, the effect of adding lysate NapA to the
supernatant wiltype on the A800 nm was
tested. For the control, the medium was used. The
absorbance was measured 24 hours after the start of the
experiment.

## **CopA**

The highest peaks have been recorded at 600 nm for CopA
(Fig. 22). However, we find that in MH broth supernatant
treated with lysate and salts, a better absorbance is
recorded compared to when MH broth nitrate is used. This
also matches the bar graphs discussed above.

![Absorption spectra for CopA samples in Mueller Hinton                           (MH) broth and MH broth nitrate.](https://static.igem.wiki/teams/4259/wiki/results/fig22.png)

**Fig. 22 |** Absorption spectra for CopA
samples in Mueller Hinton (MH) broth and MH broth
nitrate. Absorbance measurements were performed to
assess the influence of adding nitrate on the absorbance
at 800 nm (A800 nm). For all the samples,
gold and silver salts were added in the ratio 1.85:1.
Also, the effect of adding lysate CopA to the
supernatant wiltype on the A800 nm was
tested. For the control, the medium was used. The
absorbance was measured 24 hours after the start of the
experiment.

## **CueO**

As observed earlier, the best peaks are seen at 600 nm for
CueO as well (Fig. 23). However, the growth media to obtain
the supernatant seems to impact the production of
nanoparticles upon addition of lysate. The data here
revealed that MH broth nitrate may be more suitable for
CueO, given that the absorbance is higher when compared to
MH broth medium.

![bsorption spectra for NapA samples in Mueller Hinton                       (MH) broth and MH broth nitrate.](https://static.igem.wiki/teams/4259/wiki/results/fig23.png)

**Fig. 23 |** Absorption spectra for CueO
samples in Mueller Hinton (MH) broth and MH broth
nitrate. Absorbance measurements were performed to
assess the influence of adding nitrate on the absorbance
at 800 nm (A800 nm). For all the samples,
gold and silver salts were added in the ratio 1.85:1.
Also, the effect of adding lysate CueO to the
supernatant wiltype on the A800 nm was
tested. For the control, the medium was used. The
absorbance was measured 24 hours after the start of the
experiment.

Our data indicates that depending on the metal reducing
protein introduced, the nature of the supernatant may impact
the formation of nanoparticles.

## 8. DNA Cloning

![icon of a lightbulb and gearwheel](https://static.igem.wiki/teams/4259/wiki/results/goal.png)

Goal

Overexpressing the three genes *cup1*,
*copA,* and *napA* in *E. coli* for the
production of nanoparticles.

![icon of a lightbulb and gearwheel](https://static.igem.wiki/teams/4259/wiki/engineering-page/wrench.svg)

Method

Insert genes via ligation into the vector pET16b and then
transform them into *E. coli* BL21.

![icon of a lightbulb and gearwheel](https://static.igem.wiki/teams/4259/wiki/results/hypothesis.png)

Hypothesis

The transformants will lead to a higher yield and
urchin-like nanoparticles.

![icon of a lightbulb and gearwheel](https://static.igem.wiki/teams/4259/wiki/engineering-page/cap.svg)

Conclusion

Unfortunately, the ligation failed and the transformants
could not be created in the short amount of lab time the
team had.

To design our constructs we chose 3 different genes based on
literature14. The Cup1 gene from
*Candida albicans* encodes for a metallothionein, which is
a metal binding protein and was proven to be important in the
formation of nanoparticles15. The genes CopA and NapA
from *Cupriavidus metallidurans* were chosen as it was
previously shown that they are involved in the production of gold
and silver nanoparticles. The two genes were taken from
*C. metallidurans* as it is a very metal resistant strain,
especially to high concentrations of gold16.

The DNA sequences were ordered from IDT in the form of gBlocks. In
order to simplify the ligation into our vector
[pET-16b](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27parts%27%29%20%7D%7D)
the sequences were first cloned into
[pJET1.2/blunt](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27parts%27%29%20%7D%7D)
and plated on LB plates. We then isolated the plasmid by Miniprep
and digested it with two restriction enzymes BamHI and NdeI, and
checked the result by gel electrophoresis. Next, the inserts were
purified from gel and the sequences were cloned into pET-16b by
ligation with the ligase T4. The ligated product was transformed
into *E. coli* BL21 and plated on ampicillin LB plates, as
the vector contains an ampicillin resistance gene. Eventually, the
sequences were checked by diagnostic restriction digestion and
Sanger sequencing (Fig. 24-26). The sequencing results showed an
alignment for Cup1, but unfortunately the ligation for CopA and
NapA failed twice and could not be repeated due to time
constraints.

# Sequencing results

[![Schematic representation of sequence alignment for cup1](https://static.igem.wiki/teams/4259/wiki/results/fig-24.png)](https://static.igem.wiki/teams/4259/wiki/results/fig-24.png)

**Fig. 24 |** Schematic representation of
sequence alignment for *cup1*.

[![Schematic representation of sequence alignment for napA.](https://static.igem.wiki/teams/4259/wiki/results/fig25.png)](https://static.igem.wiki/teams/4259/wiki/results/fig25.png)

**Fig. 25 |** Schematic representation of
sequence alignment for *napA*.

[![Schematic representation of sequence alignment for copA.](https://static.igem.wiki/teams/4259/wiki/results/fig26.png)](https://static.igem.wiki/teams/4259/wiki/results/fig26.png)

**Fig. 26 |** Schematic representation of
sequence alignment for *copA*.

Additionally, four different genes from the ASKA collectionwere used17. The selected genes were
*copA*, *napA*, *cueO*, and *melA*.
The ASKA plasmids were isolated by Miniprep, and they were then
transformed into E. *coli* BL21. This was done to have
comparable and reproducible data to our constructs, as they are
also expressed in the BL21 strains.

In order to confirm that these genes were actually expressed, the
strains were induced with IPTG (end concentration 1mM) for 2h and
the expression was confirmed with an SDS gel (Fig. 27). As a
control the non-induced strains and BL21 were also added. The size
of the different proteins was looked up on Uniprot18.
The sizes of the proteins were 51kDA for MelA, 57kDA for CueO,
88kDA for CopA, and 93kDa for NapA. As seen on the SDS gel, the
proteins were clearly expressed.

![SDS gel of four ASKA genes CopA, NapA, CueO, and                   melA.](https://static.igem.wiki/teams/4259/wiki/results/fig27.png)

**Fig. 27 |** 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. MelE
non-induced. 9. ASKA-MelE induced with IPTG. 10. BL21 wild
type. The ladder is added as a reference.

# Supplementary

## 9. Testing the influence of ingredients of different media on nanoparticle synthesis

![icon of a lightbulb and gearwheel](https://static.igem.wiki/teams/4259/wiki/results/goal.png)

Goal

Show which components of the different media are responsible
for nanoparticle formation.

![icon of a lightbulb and gearwheel](https://static.igem.wiki/teams/4259/wiki/engineering-page/wrench.svg)

Method

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

![icon of a lightbulb and gearwheel](https://static.igem.wiki/teams/4259/wiki/results/hypothesis.png)

Hypothesis

Tryptone, beef infusion and yeast extract all contain some
form of proteins and are expected to contain reducing
agents.

![icon of a lightbulb and gearwheel](https://static.igem.wiki/teams/4259/wiki/engineering-page/cap.svg)

Conclusion

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

After the results of the experiment with 4 different media were
obtained, we noticed absorbance peaks for samples in which growth
media were used to reduce metallic salts. This was an unexpected
result, as we did not come across such a phenomenon during the
preliminary literature review. Therefore, when these absorbance
peaks were observed, we set up an experiment in which the
influence of the various media constituents on nanoparticle
formation was studied.

We tested the ingredients of LB, LB low salt, LB-Nitrate, MH and
TSBS: deionized (DI) Water, Tryptone (substitutes Casein
Hydrolysate), Yeast Extract (substitutes Soy Extract in TSBS),
Starch, NaCl, Glucose and KHPO19,20,21 (Soy Extract and
Casein Hydrolysate were substituted with ingredients that fulfill
the same function and have a comparable composition). The used
concentrations were achieved in DI water, according to the
respective recipes. We also tested the interaction between the
different constituents of MH broth, since this medium was the most
relevant for the scope of the project. We only show this plot on
this page, but the obtained spectra for all other media are shown
in
[Fig. 39-41 on the Notebook page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%20%7D%7D#fig-39). They yielded similar results.

The absorption spectra for MH, supernatant of MH broth and the
individual components of the broth, including their interactions
are shown in Fig. 28. The observed peaks at ~550 nm are only
present for tryptone-containing samples. All other compounds seem
to not have any reducing effect.

![Absorbance measurements of samples with Mueller Hinton (MH) broth ingredients and their interaction in deionized (DI) Water, with HAuCl4 and AgNO3.](https://static.igem.wiki/teams/4259/wiki/results/fig28.png)

**Fig. 28 |** Absorbance measurements of samples
with Mueller Hinton (MH) broth ingredients and their
interaction in deionized (DI) Water, with HAuCl4
and AgNO3 after 24h of incubation at room
temperature. Used conditions: [HAuCl4] = 3 mM,
[AgNO3] = 0.3 mM. MH broth composition according to
the recipe obtained from Sigma-Aldrich20.

## 10. Comparing different techniques for obtaining a cell-free system

![icon of a lightbulb and gearwheel](https://static.igem.wiki/teams/4259/wiki/results/goal.png)

Goal

Identify the most reliable method for obtaining a cell-free
system.

![icon of a lightbulb and gearwheel](https://static.igem.wiki/teams/4259/wiki/engineering-page/wrench.svg)

Method

Assess if growth can be observed after adding
Kanamycin/Chloramphenicol to the supernatant or
filter-sterilizing it.

![icon of a lightbulb and gearwheel](https://static.igem.wiki/teams/4259/wiki/results/hypothesis.png)

Hypothesis

Sterile filtering will fully eliminate growth, while
antibiotics will not eliminate all possibilities for
bacterial growth.

![icon of a lightbulb and gearwheel](https://static.igem.wiki/teams/4259/wiki/engineering-page/cap.svg)

Conclusion

Only filter sterilization eliminates all growth. Thus,
proceed in all experiments with filtered supernatants.

In order to achieve a cell-free system, it was important to ensure
that the supernatant used was free of all bacterial cells. Filter
sterilization was used to achieve this, however, it is a more
expensive process and is rather inefficient with respect to time.
Hence, this experiment was designed to check whether adding
antibiotics to the supernatant may work as a suitable alternative.

A BL21-pET16b culture having an ampicillin resistance gene, was
treated either with 20 ul of Kanamycin or Chloramphenicol. These
antibiotics are expected to kill the bacteria given they do not
have the respective resistance gene. The supernatant of the
treated BL21 cultures was then plated on LB ampicillin plates. As
a control, we added a BL21 culture, where the supernatant had been
filter sterilized. The cultures were left to grow overnight in an
incubator set to 37°C.

The image shown (Fig. 28) below indicates that supernatants
treated with antibiotics resulted in growth of bacterial colonies
overnight, whereas no growth is observed for the control.

![Agar plates showing bacterial growth after                   BL21-pET16b supernatant were treated with Kanamycin and Chloramphenicol, or                   filter sterilized.](https://static.igem.wiki/teams/4259/wiki/results/fig29.png)

**Fig. 29 |** Agar plates showing bacterial
growth after BL21-pET16b supernatant were treated with
Kanamycin and Chloramphenicol, or filter sterilized.

This data suggests that filter-sterilization is the optimal
technique to obtain a cell free system, rather than antibiotic
treatment of supernatants.

# Upscaling & Extraction

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

![icon of a lightbulb and gearwheel](https://static.igem.wiki/teams/4259/wiki/results/goal.png)

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.

![icon of a lightbulb and gearwheel](https://static.igem.wiki/teams/4259/wiki/engineering-page/wrench.svg)

Method

Set up shaking flasks with silver and gold and WT + ASKA
strains. The shaking settings are 0/20/60rpm.

![icon of a lightbulb and gearwheel](https://static.igem.wiki/teams/4259/wiki/results/hypothesis.png)

Hypothesis

Intense shaking “presses” the smaller particles together,
resulting in more agglomeration.

![icon of a lightbulb and gearwheel](https://static.igem.wiki/teams/4259/wiki/engineering-page/cap.svg)

Conclusion

Shaking has a positive effect on nanoparticle formation in
preventing agglomeration.

In this experiment the impact of shaking on nanoparticle
production was tested. In all the previous experiments the flasks
were shaken elliptically at 200 rpm. For this experiment,
*E. coli* BL21 and three different ASKA genes (CueO, NapA,
and MalE) were grown in MH broth with a gold and silver ratio of
1.85:1. These flasks were shaken in a parallel movement at three
different speeds. First, they were left on the bench without
shaking for 48h. The same strains were further tested at a
parallel shaking speed of 20 rpm and 60 rpm over the course of
48h.

The absorption spectrum was measured for all different shaking
speeds for the four different strains. *E. coli* BL21 seems
to perform best at 0 and 20 rpm, while 60 rpm seems to have the
least effect (Fig. 30). The ASKA strains MalE and NapA, show the
highest peak when not being shaken, while for CueO the highest
peak can be observed at a speed of 60 rpm (Fig.30). In general,
for the three ASKA strains the absorbance values are very close to
each other and it seems like the shaking speed does not have a
substantial effect on the yield of nanoparticles.

![ Absorption spectra of Escherichia coli BL21 and ASKA supernatant in combination with lysate of different ASKA genes at three different shaking speeds.](https://static.igem.wiki/teams/4259/wiki/results/fig30.png)

**Fig. 30 |** Absorption spectra of
*Escherichia coli* BL21 and ASKA supernatant in
combination with lysate of different ASKA genes at three
different shaking speeds. The different ASKA genes that were
tested are: MalA, NapA, and CueO. The shaking speeds that were
tested are: 0, 20, and 60 rpm.

TEM pictures were taken to see what effect the shaking has on the
morphology of the nanoparticles. The pictures were taken for all
four different strains at shaking speeds 0 rpm and 60 rpm. The
pictures for *E. coli* BL21 at a shaking speed of 0 rpm
(Fig. 31 A) show a lot of aggregates and no clear nanoparticle
formation, while at 60 rpm (Fig. 31 B) we can observe less
clumping and more distinctively sphere-like nanoparticles. The
single nanoparticles had a size around 100 nm, while the
aggregates were observed at a scale of 500 nm.

![TEM pictures of E. coli BL21 shaken at 0 and 60 rpm.](https://static.igem.wiki/teams/4259/wiki/results/fig31.png)

**Fig. 31 |** TEM pictures of
*E. coli* BL21 shaken at 0 and 60 rpm. A) Two pictures
taken of *E. coli* BL21 at 0 rpm. They show
nanoparticle aggregates and no clear nanoparticle formation.
B) Two pictures taken of *E. coli* BL21 shaken at 60
rpm. Sphere-like nanoparticles can be observed. Nanoparticles
of around 100 nm were observed.

For the ASKA strains only pictures of CueO and NapA were taken,
this was simply due to time constraints and the availability of
the TEM. ASKA-CueO shows the same behavior as BL21. At 0 rpm, we
can observe a lot of aggregates and no clear formation of
nanoparticles (Fig. 32 A), while at a shaking speed of 60 rpm the
aggregates seem to break down and we can again observe sphere-like
nanoparticles (Fig. 32 B). The single nanoparticles had a size
around 200 nm, while the aggregates were observed at a scale of
500 nm.

![TEM pictures of ASKA-CueO shaken at 0 and 60 rpm.](https://static.igem.wiki/teams/4259/wiki/results/fig32.png)

**Fig. 32 |** TEM pictures of ASKA-CueO shaken at
0 and 60 rpm. A) Two pictures taken of ASKA-CueO at 0 rpm.
They show nanoparticle aggregates and no clear nanoparticle
formation. B) Two pictures taken of ASKA-CueOshaken
at 60 rpm. Sphere-like nanoparticles can be observed.
Nanoparticles of around 200 nm were observed.

Interesting results were observed for ASKA-NapA. At 0 rpm we did
not see the agglomerates as for the 2 previous strains, but could
already see sphere-like nanoparticles (Fig. 32 A). At 60 rpm, we
could observe additional growth on the nanoparticles, indicating
the formation of spikes (Fig. 32 B). Additionally, the size
observed for both shaking speeds was around 100 nm.

![TEM pictures of ASKA-NapA shaken at 0 and 60 rpm.](https://static.igem.wiki/teams/4259/wiki/results/fig33.png)

**Fig. 33 |** TEM pictures of ASKA-NapA shaken at
0 and 60 rpm. A) Two pictures taken of ASKA-NapA at 0 rpm.
They show sphere-like nanoparticles. B) Two pictures taken of
ASKA-NapAshaken at 60 rpm. Additional growth can be
observed, indicating the formation of spikes. The size of the
nanoparticles observed was around 100 nm.

In general, shaking seems to have a
**positive effect** on the formation of
nanoparticles, especially to
**prevent agglomerates.**

## 12. Extraction of nanoparticles by size using an ultracentrifuge

![icon of a lightbulb and gearwheel](https://static.igem.wiki/teams/4259/wiki/results/goal.png)

Goal

Obtain a purified fraction of nanoparticles with a peak at
A800 nm.

![icon of a lightbulb and gearwheel](https://static.igem.wiki/teams/4259/wiki/engineering-page/wrench.svg)

Method

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

![icon of a lightbulb and gearwheel](https://static.igem.wiki/teams/4259/wiki/results/hypothesis.png)

Hypothesis

We want to isolate the nanoparticles that have an absorbance
at 800 nm.

![icon of a lightbulb and gearwheel](https://static.igem.wiki/teams/4259/wiki/engineering-page/cap.svg)

Conclusion

The nanoparticles were not separated by size. However, this
technique offers potential in the characterization of
urchin-like nanoparticles.

In this experiment the WT was used with silver and gold salts over
24h to create the nanoparticles. Furthermore, visual inspection of
the absorption spectra of the nanoparticles with different
percentages of sucrose (Fig. 34), showed a peak at 600 nm for all
samples. The height of the peak differed among the samples,
however, no correlation between the percentage of sucrose and
absorbance was observed. We were also interested in the size of
our nanoparticles, therefore we used a sucrose density gradient
ultracentrifugation technique. Our previous research findings
revealed nanoparticles of different sizes. We aimed to separate
the nanoparticles based on size using the ultracentrifuge. This is
based on the principle that the differently sized nanoparticles
would layer on the surface of a gradient whose density increases
linearly from top to bottom.

![Absorption spectra of Escherichia coli BL21                   supernatant in combination with silver and gold salts, ratio 1.85:1, and                   different percentages of sucrose.](https://static.igem.wiki/teams/4259/wiki/results/fig34.png)

**Fig. 34 |** Absorption spectra of Escherichia
coli BL21 supernatant in combination with silver and gold
salts, ratio 1.85:1, and different percentages of sucrose.

Unfortunately, after centrifugation the nanoparticles were not
separated but were all pushed to the side of the tube (Fig. 35),
making it impossible to make a conclusion about size distribution.

![Image of ultracentrifugation of biologically produced                   nanoparticles with different concentrations of sucrose. Nanoparticles were                   pushed towards the side of the tube and therefore the size distribution could                   not be determined.](https://static.igem.wiki/teams/4259/wiki/results/fig35.png)

**Fig. 35 |** Image of ultracentrifugation of
biologically produced nanoparticles with different
concentrations of sucrose. Nanoparticles were pushed towards
the side of the tube and therefore the size distribution could
not be determined.

In order to improve the outcome of this experiment a different
rotor insert is needed, called a “swing bucket rotor” that would
prevent the nanoparticles from being pushed to the sides of the
tubes. Additionally, our nanoparticles were not stabilized by a
coating and aggregates formed, this could also be a reason that
they were not properly separated in the centrifuge.

# Conclusion

With Binanox, we aimed to establish a biomanufacturing system for
the production of bimetallic nanoparticles optimized for
application in PTT. For the nanoparticles to be optimal for PTT;
an **urchin-like shape**,
**A800 nm** , and
**a size smaller than 150 nm** are desired.

The optimal conditions for nanoparticle synthesis were established
using the modeling experiments. The first iteration revealed an
optimal **gold:silver ratio of 1.85:1** and the
second iteration showed an optimal **pH of 5** and
temperature of **49 °C**. In addition,
*E.coli* BL21 supernatant showed to perform best in
**MH broth** since it yielded the best A800 nm
and TEM pictures confirmed the formation of spiky nanoparticles of
a size of around **100 nm**. These defined optimal
conditions were used in further experiments. Cell lysates
containing metal reducing proteins were introduced to the
supernatant WT, however, showed no improvement in nanoparticle
production. This could be caused by the cell lysates containing
components that interfere with protein function or nanoparticle
synthesis or by the degradation of the metal reducing proteins.
The addition of **L-DOPA** to the MelA lysate,
positively influenced the A800 nm. Furthermore, the
addition of nitrate to the MH broth seemed to not have improved
the A800 nm. Finally, our construct has not been tested
since all the transformations, except for metallothionein, failed
and due to lack of time no further experiments were performed.

For future experiments, our constructs should be tested in
combination with a protease inhibitor to prevent the degradation
of the metal reducing proteins. Furthermore, surfactants should be
added to improve the stability of the nanoparticles and prevent
the formation of aggregates. Finally, the ultracentrifuge could be
a promising technique to separate the nanoparticles based on size.
Therefore, we would like to use the ultracentrifuge in combination
with a swing bucket to prevent the nanoparticles from being pushed
toward the side.

All in all, our research findings contribute to a better
understanding into the biosynthesis of bimetallic nanoparticles
using a microbial factory. Further future experiments are required
to optimize and validate our biomanufacturing system, however the
results are promising and lay a good foundation for the
biosynthesis of nanoparticles and their application in PTT.

# References

1. Hwang, S., Nam, J., Jung, S., Song, J., Doh, H., & Kim, S.
   (2014). Gold nanoparticle-mediated photothermal therapy:
   current status and future perspective. In Nanomedicine (Vol.
   9, Issue 13, pp. 2003–2022). Future Medicine Ltd.
   https://doi.org/10.2217/nnm.14.147
2. Divya, K., Kurian, L. C., Vijayan, S., & Manakulam
   Shaikmoideen, J. (2016). Green synthesis of silver
   nanoparticles by Escherichia coli : Analysis of antibacterial
   activity. Journal of Water and Environmental Nanotechnology,
   1(1), 63–74. https://doi.org/10.7508/jwent.2016.01.008
3. Oza, G., Pandey, S., & Sharon, M. (2012). EXTRACELLULAR
   BIOSYNTHESIS OF GOLD NANOPARTICLES USING Escherichia coli AND
   DECIPHERING THE ROLE OF LACTATE-DEHYDROGENASE USING LDH KNOCK
   OUT E. coli. Journal of Atoms and Molecules; Chennai, 2(4),
   301–311. https://www.proquest.com/docview/1368017351
4. Jung, J. H., Cho, M., Seo, T. S., & Lee, S. Y. (2022).
   Biosynthesis and applications of iron oxide nanocomposites
   synthesized by recombinant Escherichia coli. Applied
   Microbiology and Biotechnology, 106(3), 1127–1137.
   https://doi.org/10.1007/s00253-022-11779-4
5. Munyayi, T. A., Vorster, B. C., & Mulder, D. W. (2022). The
   Effect of Capping Agents on Gold Nanostar Stability,
   Functionalization, and Colorimetric Biosensing Capability.
   *Nanomaterials*, *12*(14), 2470.
   https://doi.org/10.3390/nano12142470
6. 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).
7. Gurunathan, S. *et al.* Biosynthesis, purification and
   characterization of silver nanoparticles using Escherichia
   coli. *Colloids Surf B Biointerfaces*
   **74**, 328–335 (2009).
8. 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
9. Philip, P., Kern, D., Goldmanns, J., Seiler, F., Schulte, A.,
   Habicher, T., & Büchs, J. (2018). Parallel substrate supply
   and pH stabilization for optimal screening of E. coli with the
   membrane-based fed-batch shake flask. In Microbial Cell
   Factories (Vol. 17, Issue 1). Springer Science and Business
   Media LLC. https://doi.org/10.1186/s12934-018-0917-8
10. Ferrer, M., Chernikova, T. N., Yakimov, M. M., Golyshin, P.
    N., & Timmis, K. N. (2003). Chaperonins govern growth of
    Escherichia coli at low temperatures. In Nature Biotechnology
    (Vol. 21, Issue 11, pp. 1267–1267). Springer Science and
    Business Media LLC. https://doi.org/10.1038/nbt1103-1266b
11. Khodashenas, B. (2015). Nitrate reductase enzyme in
    Escherichia coli and its relationship with the synthesis of
    silver nanoparticles.
    *UCT Journal of Research in Science, Engineering and
    Technology*, *3*(1), 26-32.
12. Tsai, Y. J., Ouyang, C. Y., Ma, S. Y., Tsai, D. Y., Tseng, H.
    W., & Yeh, Y. C. (2014). Biosynthesis and display of diverse
    metal nanoparticles by recombinant Escherichia coli.
    *RSC Advances*, *4*(102), 58717-58719.
13. Rensing, C., & Grass, G. (2003). Escherichia coli mechanisms
    of copper homeostasis in a changing environment.
    *FEMS microbiology reviews*, *27*(2-3), 197-213.
14. Chen, A., Keitz, B. K., & Contreras, L. M. (2018). Biological
    links between nanoparticle biosynthesis and stress responses
    in bacteria. *Mexican journal of biotechnology*,
    *3*(4), 44-69
15. Adamo, G. M., Lotti, M., Tamás, M. J. & Brocca, S.
    Amplification of the CUP1 gene is associated with evolution of
    copper tolerance in Saccharomyces cerevisiae.
    *Microbiology (United Kingdom)* **158**,
    2325–2335 (2012).
16. Lal, D., Nayyar, N., Kohli, P., & Lal, R. (2013, March).
    Cupriavidus metallidurans: A modern alchemist. Indian journal
    of microbiology. Retrieved September 21, 2022, from
    https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3587520/
17. Kitagawa, M., Inamoto, E., Ioko, T., Arifuzzaman, M., & Ara,
    T. (n.d.).
    *Complete set of ORF clones of escherichia coli ASKA library
    (a complete set of E. Coli K-12 ORF archive): Unique
    Resources for Biological Research*. DNA research : an international journal for rapid
    publication of reports on genes and genomes. Retrieved
    September 21, 2022, from
    https://pubmed.ncbi.nlm.nih.gov/16769691/
18. Bateman, A. *et al.* UniProt: the universal protein
    knowledgebase in 2021. *Nucleic Acids Res*
    **49**, D480–D489 (2021).
19. Green, M. R., & Sambrook, J. (2012).
    *Molecular Cloning: A Laboratory Manual (Fourth Edition):
    Three-volume set*
    (4th ed.). Cold Spring Harbor Laboratory Press.
20. Sigma-Aldrich. (2018). 70192 Mueller Hinton Broth (M-H Broth).
    Retrieved September 21, 2022
21. 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
    e-tryptic-soy-broth

{% endblock %}