iGEM 2022 — Team nanjing-china

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## January

8th January: We finalized our team members as well as crews for every group. And we had our first plenary meeting face-to-face.

## February

27th February: In a team meeting with our PI and advisors, we had a brainstorm and finalized the MFC as our project idea.

## March

1st March: We studied the criterion of the previous iGEM competition and shared ideas with each other.

24th Marth: The group responsible for the experiment and modeling performed a literature search for the principle of MFC and relevant experiment operation and models of Shewanella.

## April

5th April: The team registration on the official website of iGEM.

11th April: We did some research to help design and build biobricks for AgBP2. And we cultured the *E.coli* and *Shewanella oneidensis* in the media of LB for the following experiment.

12th April: We referred to related research to establish a growth model of Shewanella under the condition of silver ions.

15th - 17th April: Attempted to get recon of the plasmid containing the sequence of AgBP2 by PCR and MultiS One Step Cloning Kit.

19th - 21st April: The second attempt to get recon of the plasmid containing the sequence of AgBP2.

23rd - 26th April: Conducted the transformation experiment of AgBP2 for the third time and screened to get the recon *E. coli*.

29th April: Had a meeting to discuss the progress of the experiment in the last three weeks.

## May

2nd May: The team co-organized the online iGEM Meeting up with teams XJTLU-CHINA, ZJU-China, ZJUT China, and CPU\_CHINA. We had the desire to build a partnership with team XJTLU-CHINA.

5th - 7th May: Separated the plasmid containing the sequence of AgBP2 from the recons and sequenced the plasmids. Cultured the Shewanella for the following electrotransformation.

8th May: Attempted to get Shewanella recon containing the AgBP2 fragment by electrotransformation. Screened the recons.

12th May: The group responsible for the experiment performed a literature search and shared ideas about the construction of MFC.

13th May: Repeated the eletrotransformation experiment of Shewanella.

17th - 21st May: Carried out the eletrotransformation experiment of Shewanella for the third time. However, we still failed to get the recon containing Atox1 by LB with Kanamycin. We decided to redesign our experiment.

18th May: The group responsible for the experiment met Professor Zhang Jianrong and consulted the details and information of the electrochemical workstation.

22nd May: We had a team meeting to summarize the work in the past few months. The group responsible for the experiment made a presentation about the principle as well as the progress of the transformation of E.coli and Shewanella.

## June

1st June: Exchanged ideas with team XJTLU-CHINA, who were carrying out a similar project about Ag+ at that time. They taught us to adjust the voltage intensity continuously in each repeat of eletrotransformation.

2nd June: Our instructor encouraged us to try another silver-binding protein, Atox1(204bp), which is a larger silver-binding protein than AgBP2(36bp).

6th - 8th June: Designed and built biobricks for Atox1. And cultured the E.coli and Shewanella oneidensis in the media of LB for the following experiment.

13th - 15th June: Attempted to get recon of the plasmid containing the sequence of Atox1 by PCR and MultiS One Step Cloning Kit.

16th - 18th June: Repeated the transformation experiment of Atox1.

20th - 23rd June: Screened and got the E.coli recon containing the Atox1 fragment. Scaled up the culture of recon.

27th June: 30th June: Separated the plasmids containing a sequence of Atox1 from the recons and sequenced the plasmids. Cultured the Shewanella for the following electrotransformation.

## July

11th July: Had a team meeting to summarize the HP and modeling progress. We also discussed the details of our project direction.

13th July: Collaboration meeting with team XJTLU. Talked about project direction and possible collaboration.

14th - 16th July: Attempted to get Shewanella recon containing the Atox1 fragment by electrotransformation. Screened the recons.

18th - 20th July: Repeated the eletrotransformation experiment to transform the Atox1 into the Shewanella, during which we adjusted the voltage intensity continuously in each repeat.

22nd - 24th July: Improved and repeated the eletrotransformation experiment of Shewanella for the third time. Screened and got the recon containing Atox1 by LB with Kanamycin.

27th July: We attended the online Fresh Freshwater Bioremediation Alliance Meetup and made a presentation for our project.

30th July: Prepared the anode materials and devices for the three-electrode cell experiment. Cultured the Shewanella with Atox1 as the experimental group and wild Shewanella as a control group.

## August

5th August: Started the three-electrode cell experiment and recorded the output current.

19th August: We attended the Conference of China iGEMer Community(CCiC) and made a presentation for our project.

20th August: Had a discussion to finalize the pattern of our team uniform.

26th August： We visited the Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Science, and interviewed Dr. Cong.
31st August: We had a meeting to summarize the progress of HP, MFC construction, modeling, and wiki writing with our PI and Advisor.

## September

2nd September: Collaboration meeting with team XJTLU-CHINA to discuss and share possible measures of Biosafety in our project when it is applied in the real world.

24th September： We gave an online lecture about Synthetic biology and the future of humans to the students in Shuangbai No.1 middle school.

25th September: We referred to related research to establish a simulation model of microbial fuel cells under lactic acid condition

## October

7nd October: We add new documentation to an existing Part, pCusC mKate2 (BBa\_K1980007), and expand its applications in silver ions indicator through paper reading and our experiment results.We detected the fluorescence of transfermed E. coli at different concentrations of silver ions.

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Engineering success is always a very important part of both iGEM and synthetic biology. Design–Build–Test–Learn (DBTL) cycle is traditional engineering guidance for synthetic biology. Following this principle, our iGEM project carried out a series of experiments and researches to achieve engineering success.

## Engineering

### Design

MFC can directly convert chemical energy stored in organic matter into electricity and have important applications in power generation and wastewater treatment. However, current MFCs generally exhibit undesirably low power density. So how to improve the power density?

With this question, we interviewed an investor in the field of biotechnology, who knows a lot about electrogenic microorganisms. He told us that MFC has three main limiting factors: electrodes, device design, and strains. And for strains, the sluggish transmembrane and extracellular electron-transfer processes are the biggest flaws. After brainstorming, we decided to solve this problem with synthetic biology.

![](https://static.igem.wiki/teams/4134/wiki/wiki/engineering/01guolichuan.jpg)

Introducing Shewanella transmembrane and outer-membrane silver nanoparticles can boost the charge-extraction efficiency, reports a study published in 2021 in Science. They added chemically synthesized AgNPs to electrodes and their MFC reached the highest current density ever. This research aroused our interest in AgNPs. We plan to display a silver-binding protein on the membrane of Shewanella to help it capture silver ions and reduce silver into nanoparticles so as to boost charge-extraction efficiency in Shewanella microbial fuel cells.

### Built

In our project, silver is a double-edged sword. It can both increase the current density of bacteria and kill bacteria. We need to consider the proper silver concentration. So, we started to design our first model.

We constructed a Suppressed-Active-Dead (SAD) Model according to the research of Haque et al. This model describes the inhibition of E. coli by silver ions. This model helped us better understand our work. After demonstrating that the model was widely available, we used it to estimate the amount of silver ions in the pre-experiment and help us narrow down silver concentrations and built a concentration gradient. See more in [Model](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D)

### Test

We carried out wet lab experiments and proved that silver ions of 10μmol/L and below do not harm the growth of Shewanella cells. However, the results do not agree with the model at higher silver ion concentrations.

![](https://static.igem.wiki/teams/4134/wiki/wiki/engineering/02-pre-experiment.jpg)

### Learn

We learned that we need to model the growth curve according to the conditions under which we grow bacteria. We revised the model parameters based on experimental data to make the model more accurate.

![](https://static.igem.wiki/teams/4134/wiki/wiki/engineering/03-rebuild.jpg)

![](https://static.igem.wiki/teams/4134/wiki/wiki/engineering/04-cycle-1.jpg)

### Design

For the MFC design, we plan to add a small amount of silver nitrate to the anode chamber. We display silver-binding protein on the C-terminal of BpfA, a large membrane protein of \_Shewanella\_. Silver ions are captured on the membranes and then reduced \_in situ\_ by cellular metabolism-generated electrons into the transmembrane silver nanoparticles inside and traversing the cell membranes. Generally, electrons are transferred between Fe3+/Fe2+ redox centers through a multi-step hopping process. Since the electronic conductivity of metals is much higher than the redox center-mediated charge transfer process in cytochromes, transmembrane and outer membrane silver nanoparticles may act as metal shortcuts to bypass redox center-mediated slow electrons(Cao B, Science,2021). During the transfer process, it is in direct contact with the external electrode to extract the charge more efficiently and improve the current density of the MFC.

![](https://static.igem.wiki/teams/4134/wiki/wiki/engineering/05-membrane.jpg)

### Built

To realize the fusion expression of silver-binding protein and Shewanella membrane protein, we built a new recombination device. It includes BpfA, the Carbon-terminal 1000bp sequence of a large membrane protein; AgBP2, a silver binding protein; Kanamycine resistance to select recombinants; AggC, the Nitrogen-terminal sequence for the downstream gene of BpfA.

When the device was transferred into Shewanella, the silver-binding protein could recombine to the Carbon-terminal of BpfA for silver in situ reductions.

![](https://static.igem.wiki/teams/4134/wiki/wiki/engineering/06-recombination-device-1.jpg)

### Test

In the first round of experiments, we used AgBP2 as a silver-binding protein and designed our homologous recombination device, and built the plasmid to amplify the device. However, the electrotransformation in 1.8kV/mm failed a few times. The clone that can grow on a plate with kanamycin is our target recombinant but we didn’t see any of them.

### Learn

From those failures, we learned that the electrotransformation of Shewanella is relatively difficult. So we sought help from our partner, XJTLU-China, and they advised us to adjust the voltage intensity of electrotransformation continuously. And our adviser encouraged us to try another silver-binding protein, Atox1(204bp), which is a larger silver-binding protein than AgBP2(36bp).

![](https://static.igem.wiki/teams/4134/wiki/wiki/engineering/07-cycle-2.jpg)

### Redesign and Built

We redesigned and built our new device with Atox1, then carried out electrotransformation at 1.2 kilovolts per millimeter. Finally, we get a qualified recombinant.

![](https://static.igem.wiki/teams/4134/wiki/wiki/engineering/08-recombination-device-2.jpg)

### Test

* First, this recombinant clone can grow on a plate with kanamycin.
* Furthermore, we carried out silver nanoparticle tests and half-cell current tests.
* The ability to produce silver nanoparticles was proved by TEM, UV-visible Spectroscopy, EDX, and XPS results.
* The improvement of MFC current density is proved by half-cell experiments in comparison.

The results of TEM show that silver nanoparticles were obtained on the cell surface. Compared with the wild type,  the nanoparticle of our strain has a smaller size and higher density. EDX and XPS results confirm those nanoparticles are silver in the correct valence.

In the half-cell experiment, we built a three-electrode system and designed four working electrodes. It's worth noting that we added chemically synthesized nano-silver particles and bio-synthesized ones as we designed. The I-t curve was measured at a constant voltage of 0.2V. The electrode with our stains obtains a higher peak current than the ordinary electrode. In addition, comparing the chemically synthesized silver nanoparticles with the biosynthetic ones (add 1mM Ag+), we use only 5% as much silver as is used in our reference to achieve a similar current intensity.

![**The edited strain (the electrode without nano-silver) is improved.** The concentration of working fluid (\(\ce{AgNO3}\)) is 1mM, only 5% of that of the electrode with nano-silver.](https://static.igem.wiki/teams/4134/wiki/wiki/results/18.png)

### Learn

We confirm that we built the strain as we designed. The bio-synthesized silver nanoparticles could boost charge-extraction efficiency in Shewanella microbial fuel cells.

![](https://static.igem.wiki/teams/4134/wiki/wiki/engineering/09-cycle-3.jpg)

**To date, we finished the three Design-Build–Test–Learn cycles, which is helpful for our final success throughout the whole program.**

For detailed experiment results, you can check on [Results page](https://2022.igem.wiki/nanjing-china/results)

## References 1. Li, X.-Y., Zhou, X.-D. & Hu, J.-M. A straightforward and reliable evaluation of Ag(I) binding affinity mediated by a peptide ligand for constructing an efficient sensing platform. Talanta 226, 122089 (2021). 2. Cao, Y. et al. A Synthetic Plasmid Toolkit for Shewanella oneidensis MR-1. Front. Microbiol. 10, 410 (2019). 3. Sun, P., Li, K., Lin, K., Wei, W. & Zhao, J. Ag 2 S Quantum Dots for Use in Whole-Cell Biohybrid Catalyst for Visible-Light-Driven Photocatalytic Organic Pollutant Degradation. ACS Appl. Nano Mater. 5, 9754–9760 (2022). 4. Wang, X. et al. An unexpected all-metal aromatic tetranuclear silver cluster in human copper chaperone Atox1. Chem. Sci. 13, 7269–7275 (2022). 5. Ge, C. et al. Biotic Process Dominated the Uptake and Transformation of Ag + by Shewanella oneidensis MR-1. Environ. Sci. Technol. 56, 2366–2377 (2022). 6. Hall Sedlak, R. et al. Engineered Escherichia coli Silver-Binding Periplasmic Protein That Promotes Silver Tolerance. Appl Environ Microbiol 78, 2289–2296 (2012). 7. Lin, T. et al. Engineered Shewanella oneidensis-reduced graphene oxide biohybrid with enhanced biosynthesis and transport of flavins enabled the highest bioelectricity output in microbial fuel cells. Nano Energy 50, 639–648 (2018). 8. Bai, X. et al. Engineering synthetic microbial consortium for efficient conversion of lactate from glucose and xylose to generate electricity. Biochemical Engineering Journal 172, 108052 (2021). 9. Yang, Y. et al. Enhancing Bidirectional Electron Transfer of Shewanella oneidensis by a Synthetic Flavin Pathway. ACS Synth. Biol. 4, 815–823 (2015). 10. Song, R.-B. et al. Graphene/Fe 3 O 4 Nanocomposites as Efficient Anodes to Boost the Lifetime and Current Output of Microbial Fuel Cells. Chem. Asian J. 12, 308–313 (2017). 11. Zou, L., Huang, Y., Long, Z. & Qiao, Y. On-going applications of Shewanella species in microbial electrochemical system for bioenergy, bioremediation and biosensing. World J Microbiol Biotechnol 35, 9 (2019). 12. Zhou, G., Yuan, J. & Gao, H. Regulation of biofilm formation by BpfA, BpfD, and BpfG in Shewanella oneidensis. Front. Microbiol. 6, (2015). 13. Cao, B. et al. Silver nanoparticles boost charge-extraction efficiency in Shewanella microbial fuel cells. Science 373, 1336–1340 (2021). 14. Sivakumar, K. et al. Surface display of roGFP for monitoring redox status of extracellular microenvironments in Shewanella oneidensis biofilms. Biotechnol. Bioeng. 112, 512–520 (2015). 15. Gudipaty, S. A. & McEvoy, M. M. The histidine kinase CusS senses silver ions through direct binding by its sensor domain. Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics 1844, 1656–1661 (2014).

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## PCR

### Supplies

* 2×Phanta Flash Master Mix
* Bio-rad T100 Thermal Cycler
* PCR tubes

### Materials

|  | Atox1 | Agbp2 | aggC | bpfA |
| --- | --- | --- | --- | --- |
| Forward primer | √ | √ | √ | √ |
| Reverse primer | √ | √ | √ | √ |
| Template DNA | √ | √ | √ | √ |

* \(\ce{ddH2O}\)

### Protocol

#### PCR of Atox1, BpfA, AggC

1. In 3 PCR tubes, mix in the following order on ice :

| Reagent | Volume |
| --- | --- |
| Forward primer of Atox1 | 2μl |
| Reverse primer of Atox1 | 2μl |
| 2×Phanta Flash Master Mix | 25μl |
| Template DNA of Atox1 | 5μl |
| \(\ce{ddH2O}\) | 15μl |

2. In 3 PCR tubes, mix in the following order on ice :

| Reagent | Volume |
| --- | --- |
| Forward primer of bpfA | 2μl |
| Reverse primer of bpfA | 2μl |
| 2×Phanta Flash Master Mix | 25μl |
| Template DNA of bpfA | 5μl |
| \(\ce{ddH2O}\) | 15μl |

3. In 3 PCR tubes, mix in the following order on ice :

| Reagent | Volume |
| --- | --- |
| Forward primer of aggC | 2μl |
| Reverse primer of aggC | 2μl |
| 2×Phanta Flash Master Mix | 25μl |
| Template DNA of aggC | 5μl |
| \(\ce{ddH2O}\) | 15μl |

The total volume of reagent assembled in each of the PCR tubes is 50μl.

4. Place the 9 tubes into the thermal cycler and close the lid.
5. Amplify the acids using the denaturation, annealing, and polymerization times and temperatures listed below.

| Temperature | Time | Cycle Number |
| --- | --- | --- |
| 98℃ | 10 sec | 30 cycles |
| 58℃(\(\mathrm{T\_m}\)) | 5 sec |
| 72℃ | 5 sec |

6. Withdraw 5μl from the PCR product and analyze them by agarose gel electrophoresis.

#### PCR of Agbp2,bpfA,aggC

Same with the PCR protocol of Atox1,bpfA,aggC.

## ClonExpress MultiS

### Material

* Template for fragment amplification
* Primer
* Linearization carrier
* pfu ultra: Phanta® Max Super-Fidelity DNA Polymerase
* Competent cell
* \(\ce{ddH2O}\)
* PCR Tubes
* PCR Amplifier

### Protocol

1. Preparation of linearized carrier

* The appropriate cloning site was selected and the vector was linearized. It is recommended that the region with no repetitive sequence and uniform GC content be selected as far as possible. When the GC content in the upstream and downstream 20bp region of the vector cloning site is between 40% Mel 60%, the recombination efficiency will reach the maximum.
* Vector linearization: you can choose restriction endonuclease digestion or reverse PCR amplification.

1. Insert fragment to get

* By introducing the homologous sequence at the 5' end of the primer, there are identical sequences (15-20bp, excluding restriction sites) among the amplification products and between the amplification products and the linearized cloning vector.
* The inserted fragment was amplified by PCR.

1. Usage of linearized vectors and inserts

* Concentration determination
* Calculation of the usage of carriers and fragments

1. Recombination reaction

* The amount of DNA needed for the recombination reaction was calculated according to the formula. In order to ensure the accuracy of the sample addition, the linearized vector and the insert can be diluted properly before preparing the recombination system, and the sample volume of each component is not less than 1 μl.
* Prepare the following reaction system on ice

| Constituent | Recombination reaction | Negative control-\(1^b\) | Negative control-\(2^c\) | Positive control\(^d\) |
| --- | --- | --- | --- | --- |
| Linearized vector\(^a\) | X μl | X μl | 0 μl | 1 μl |
| n inserts\(^a\) | Y\(\_1\) - Y\(\_n\) μl | 0 μl | Y\(\_1\) - Y\(\_n\) μl | 1 μl |
| 5×CE MultiS Buffer | 4 μl | 0 μl | 0 μl | 4 μl |
| Exnase MultiS | 2 μl | 0 μl | 0 μl | 2 μl |
| \(\ce{ddH2O}\) | to 20 μl | to 20 μl | to 20 μl | to 20 μl |

* Use the pipette to gently suck and mix well (please do not oscillate and mix well), and briefly centrifuge to collect the reaction solution to the bottom of the tube.
* Reaction at 37 ℃ for 30 min; drop to 4 ℃ or immediately cool on ice.

1. Transformation of recombinant product

* Thaw chemical competent cells for cloning on ice.
* Add 10 μl recombinant product to 100 μl competent cells, mix the tube wall evenly (please do not shake and mix well), and place 30min on the ice.
* After being heated in a water bath at 42 ℃ for 45 sec, it was immediately placed on ice to cool for 2-3 min.
* Add 900ul SOC or LB medium (without antibiotics) and shake bacteria at 37 ℃ for 1 h (rotation speed 200-250rpm).
* The corresponding resistant LB plate solid medium was preheated in 37 ℃ incubator.
* The supernatant of 900 μl was discarded by 5000rpm centrifugation 5min. The bacteria were re-suspended with the remaining medium and gently smeared evenly on the plate with correct resistance with a sterile coating stick.
* The cells were cultured upside down in an incubator at 37 ℃ for 12-16 h.

1. Identification of recombinant products

## Agarose Gel Electrophoresis

### Supplies

* SUb-Cell GT Agarose Gel Electrophoresis System
* ChemiDoc Touch Gel Imaging System
* Microwave oven
* Electronic scale
* 200ml erlenmeyer flask
* 50ml and 1000ml graduated cylinders
* Power source

### Materials

* Agarose
* GelRed nucleic acid dyes
* DL2000 DNA Marker
* DL5000 DNA Marker
* PCR products
* Double digestion products

### Protocol

#### Agarose gel electrophoresis of PCR products

1. Add 1.2g of agarose and 80ml of 1×TAE buffer to a Erlenmeyer flask,and swirl to suspend the agarose powder in the buffer.
2. Place the gel solution into the microwave.Using a low to medium setting,set the timer for a minimum of 5 minutes,stopping the microwave oven every 30 seconds and swirling the flask gently to suspend the undissloved agarose.
3. Boil and swirl the solution until all of the small translucent agarose particles are dissolved.
4. Set aside the flask to cool to 60℃ before adding 2μl GelRed nucleic acid dyes.
5. Place the comb into the 15×7 cm slot of the tray and pour the molten agarose into the gel tray.
6. Allow 30-40 minutes for the gel to solidify at room temperature.
7. Carefully remove the comb from the solidified gel and remove the tape from the edges of the gel tray.Place the tray onto the leveled Sub-Cell so that the sample wells are near the cathode.
8. Submerge the gel beneath 2 to 6 mm of 1× TAE buffer.
9. Withdraw 2.5μl from the PCR products as loading samples. Load the samples as the following arrangement:

| Sample well number | 1,7 | 2-4 | 5,6,8 | 9-11 |
| --- | --- | --- | --- | --- |
| Sample type | DL2000 DNA Marker | bpfA | aggC | Agbp2/Atox1 |
| Volume | 8μl | 2μl | 2μl | 2μl |

10. Close the lid of gel tank and apply a voltage of 75V.
11. When the DNA samples or dyes have migrated a suffcient distance through the gel,turn off the electric current and remove the leads and lid from the gel tank.
12. Place the gel on the UV transilluminator for nucleic acid visualization and analysis.

#### Agarose gel electrophoresis of double digestion products

1. Add 1.2g of agarose and 80ml of 1×TAE buffer to a Erlenmeyer flask,and swirl to suspend the agarose powder in the buffer.
2. Place the gel solution into the microwave.Using a low to medium setting,set the timer for a minimum of 5 minutes,stopping the microwave oven every 30 seconds and swirling the flask gently to suspend the undissloved agarose.
3. Boil and swirl the solution until all of the small translucent agarose particles are dissolved.
4. Set aside the flask to cool to 60℃ before adding 2μl GelRed nucleic acid dyes.
5. Place the comb into the 15×7 cm slot of the tray and pour the molten agarose into the gel tray.
6. Allow 30-40 minutes for the gel to solidify at room temperature.
7. Carefully remove the comb from the solidified gel and remove the tape from the edges of the gel tray.Place the tray onto the leveled Sub-Cell so that the sample wells are near the cathode.
8. Submerge the gel beneath 2 to 6 mm of 1× TAE buffer.
9. Withdraw 2.5μl from the PCR products as loading samples. Load the samples as the following arrangement:

| Sample well number | 1 | 2-11 |
| --- | --- | --- |
| Sample type | DL5000 DNA Marker | double digestion products |
| Volume | 8μl | 50μl |

10. Close the lid of gel tank and apply a voltage of 85V for nearly 50 minutes.
11. When the DNA samples or dyes have migrated a suffient distance through the gel,turn off the electric current and remove the leads and lid from the gel tank.
12. Place the gel on the UV transilluminator for nucleic acid visualization and analysis.

## Extract the plasmid

### Material

* Bacterial solution
* Buffer P1
* Buffer P2
* Buffer P3
* FastPure DNA Mini Columns
* Collection Tube 2 ml
* Buffer PW1
* Buffer PW2
* Elution Buffer

### Protocol

1. Take 1-5 ml overnight cultured bacterial solution, add it to a centrifuge tube, and centrifuge at 10,000 rpm for 1 min. Discard the medium and invert it on absorbent paper to absorb the residual liquid.
2. Add 250 μl of Buffer P1 to the centrifuge tube with the bacterial pellet left, and mix with a pipette or vortex.
3. Add 250 μl of Buffer P2, and gently mix up and down 8-10 times to fully lyse the cells.
4. Add 350 μl of Buffer P3, and immediately gently invert 8 - 10 times to completely neutralize Buffer P2. At this point, a white flocculent precipitate should appear. Centrifuge at 12,000 rpm for 10 min.
5. Place the FastPure DNA Mini Columns in a Collection Tube 2 ml collection tube. Transfer the supernatant carefully to the adsorption column with a pipette, taking care not to aspirate the pellet, and centrifuge at 12,000 rpm for 30 - 60 sec. Discard the waste liquid in the collection tube and put the adsorption column back into the collection tube.
6. Add 500 μl Buffer PW1 to the adsorption column. Centrifuge at 12000 rpm for 30-60 sec. abandoned waste liquid, put the adsorption column back into the collection tube.
7. Add 600 μl Buffer PW2 (diluted with absolute ethanol) to the adsorption column. 12000rpm centrifuge for 30 - 60 sec. Discard the waste liquid and put the adsorption column back into the collection tube.
8. Repeat the previous step
9. Put the adsorption column back into the collection tube. Dry the adsorption column by centrifugation at 12,000 rpm for 1 min to completely remove the residual rinse solution in the adsorption column.
10. Place the adsorbent in a new sterile 1.5 ml centrifuge tube. Add 30-100 ul Elution Buffer to the center of the membrane of the column adsorption column. Incubate at room temperature for 2 min, and centrifuge at 12,000 rpm for 1 min to elute DNA.
11. The adsorption column was discarded and the DNA product was stored at -20°C to prevent DNA degradation.

## Double enzyme digestion - EcoR I and Kpn I

### Material

* DNA
* QuickCut Kpn I
* QuickCut EcoR I
* 10X QuickCut Buffer
* 10X QuickCut Green Buffer
* Enzyme storage solution
* Sterilized water

### Protocol

1. Prepare the reaction solution according to the following table:

|  | Linear DNA | Plasmid DNA | PCR Product |
| --- | --- | --- | --- |
| 10X QuickCut Buffer or 10X QuickCut Green Buffer\* | 1 μl-5 μl | 1 μl-5 μl | 1 μl-3 μl |
| DNA | ≤1 μg | ≤1 μg | ≤0.2 μg |
| QuickCut EcoR I | 1 μl | 1 μl | 1 μl |
| QuickCut Kpn I | 1 μl | 1 μl | 1 μl |
| Sterilized water | Up to 10 μl-50 μl | Up to 10 μl-50 μl | Up to 10 μl-30 μl |

2. Mix gently and centrifuge instantly.
3. Heat preservation at 37 ℃ for 5 min.

## Preparation of competent Shewanella

### Materials

* Sucrose
* 1.5mL microfuge tubes
* The *S. oneidensis* MR-1 cultured overnight
* \(\ce{ddH2O}\)

### Protocol

1. Prepare 300mM sucrose solution by dissolving 4.11g of sucrose in 40ml of \(\ce{ddH2O}\).
2. Add 1.5ml of cell suspension into each 1.5ml microfuge tube.
3. Centrifuge at a speed of 10000rpm for 2 minutes.
4. Remove the supernatant by aspiration.
5. Add 1ml of sucrose solution into each microfuge tube to suspend the cells gently.
6. Repeat the steps 3,4 for 2 times.
7. Centrifuge the cell suspension at a speed of 10000rpm for 2 minutes.
8. Remove the supernatant by aspiration.
9. Add 100μl of sucrose solution into the center of the microfuge tube and suspend the cells gently.

## Electroformation

### Supplies

* MicroPulser Electroporator
* MicroPulser Electroporation cuvettes(0.1cm)
* 2ml centrifuge tubes
* Thermostatic water bath cauldron(30℃)

### Materials

* LB medium
* LB agar with Kanamycin(20μg/L)
* Competent *S. oneidensis* MR-1
* Plasmid DNA

### Protocol

1. Add 1ml of LB medium into a 2ml centrifuge tube and put it into a 30℃ water bath cauldron.
2. Turn the electroporator and set up the voltage to 1.2kv.
3. Transfer 100μl of competent cell suspension and DNA(approximately 2.4μg) to a cold electroporation cuvette by micropipette.
4. Place the cuvette in the chamber slide. And push the slide into the chamber until the cuvette is seated between the contacts in the base of the chamber.
5. Press the "Pulse" button to complete a pulse.
6. Remove the cuvette from the chamber and immediately add 1mL of LB medium to the cuvette. Gently resuspend the cells.
7. Transfer the cell suspension to a 2ml centrifuge tube and incubate at 30℃ for 2 hr.
8. Centrifuge at 10000rpm.
9. Plate on the LB agar with Kanamycin(20μg/L) and incubate at 30℃.

## Biofilm formation assay

### Supplies

* 96-well plate
* Microplate reader

### Materials

* *S.oneidensis* (wild type[WT])
* *S.oneidensis* (mutant)
* Crystal violet(1% aqueous solution)
* Crystal violet(1% aqueous solution)
* LB medium
* MM1 medium

| Component | Concentration |
| --- | --- |
| Hepes | 30mM |
| NaOH | 7.5mM |
| \(\ce{NH4Cl}\) | 28.04mM |
| KCl | 1.34mM |
| \(\ce{NaH2PO4}\) | 4.35mM |
| \(\ce{CaCl2}\) | 0.68mM |
| Add trace amounts of minerals, vitamins, and amino acids(pH~7.0). |  |

### Protocol

1. Mix about 40μl of the overnight LB cultures(OD600~1.0) with 200μl MM1 medium on a 96-well plate.
2. Incubate the cells at 30℃ for 24 or 48 hours.
3. Discard the planktonic cells.
4. Add 100μl of crystal violet to each well. Strain the surface-associated cells for 10 minutes.
5. Remove the excess CV from each well and dry the plate at room temperature for 40 minutes.
6. Add 100μl of 95% ethanol into each well for 10 minutes to resuspend the cells.
7. Quantify the amount of biofilm biomass in each well by measuring the OD260 in the Microplate reader.

## Half-cell microbial fuel cell experiment

### Material

* LB Liquid medium
* *Shewanella* recombinant
* Ammonium lactate
* Silver nitrate
* Three-electrode system:
* Working electrode: Carbon paper, Carbon paper-rGO, Carbon paper-rGO-AgNPs (chemical synthesis), Carbon paper-rGO-AgNPs (biosynthesis in this study)
* Counter electrode: platinum electrode
* Reference electrode: Ag/AgCl
* M9 buffer

| Ingredients | Concentration |
| --- | --- |
| \(\ce{Na2HPO4}\) | 6g/L |
| \(\ce{KH2PO4}\) | 3g/L |
| NaCl | 0.5g/L |
| \(\ce{NH4Cl}\) | 1g/L |
| \(\ce{MgSO4·7H2O}\) | 0.247g/L |
| \(\ce{CaCl2·2H2O}\) | 0.0147g/L |

### Protocol

1. The recombinant *Shewanella* selected after electroporation was added to 10 ml LB liquid medium for overnight culture for 12 hours.
2. The 600nm absorbance (OD600) of the bacterial liquid was determined, and the value was recorded as x, and the bacterial liquid with a volume of 40 ml\*1.2/x was taken from the centrifuge tube. (the bacterial liquid concentration is expressed by optical density and defined as the common logarithm of the ratio of incident light intensity to transmitted light intensity, OD=lg (1/trans), where trans is the light transmission value of the detector).
3. The bacterial solution was centrifuged with 7000-8000 rpm 2 min, and the supernatant was taken and re-suspended with appropriate amount of M9 buffer.
4. Repeat step 3 1 to 2 times for washing precipitation.
5. To prepare the working solution of 40 ml electrolytic cell: add 2 ml LB culture solution to the bacterial liquid obtained from the last re-suspension, fill the volume to 40 ml with M9, add sodium lactate to 19 mmol/L (40 ml additive 85mg ammonium lactate), and use nitrogen 15min before use to remove dissolved oxygen.
6. Set up a three-electrode test device and prepare four different electrodes, in which the experimental group needs to add silver nitrate (1 mmol/L) to the working solution.
7. The current intensity was measured at a constant voltage of 0.2 V and the I-t curve was recorded.

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## 1. The Tolerance of Ag+

According to the general range of the concentration of \(\ce{Ag+}\) (1～40μM) obtained by the SAD model, different gradients of the solid medium are prepared. The same number of Shewanella are inoculated in each group, and the growth of it at different concentrations of \(\ce{Ag+}\) is observed (figure1). The results are more obvious when the number of inoculated Shewanella is small. From the point of view of the number of colonies, the activity of Shewanella decreases with the increase of the concentration of \(\ce{Ag+}\).

![Figure1. **The CFU of all of the experimental groups (Atox1) are less than the control groups.**For each figure, the left line represents the control group, and the right line represents the experimental group (Atox1). The number of Shewanella is \\(5.0\cdot 10^{5}\\), \\(5.0\cdot 10^{4}\\), \\(5.0\cdot 10^{3}\\), and \\(5.0\cdot 10^{2}\\) respectively from top to bottom. Every circular area shows the colony forming units of the Shewanella. And the four figures represent different concentrations of \\(\ce{AgNO3}\\) solution, of 0 μM, 10 μM, 20 μM and 40 μM respectively from left to right. As the concentration of \\(\ce{AgNO3}\\) solution increases progressively, the colony forming units per milliliter decreases correspondingly.](https://static.igem.wiki/teams/4134/wiki/wiki/results/01-silver-tolerantcut.jpg)

We tested the growth density of shewanella under different concentrations of \(\ce{Ag+}\) in 12 hours. We focus on concentrations of 10 μM and higher. The density of SW-MR-Atox1 is higher than that of the SW-MR-1, but when there is kanamycin, the density decreases, which means that kanamycin has some negative influence on SW-MR-Atox1.(figure2)

![Figure2. <strong>The density of SW-MR-Atox1 is higher than that of the SW-MR-1, but when there is kanamycin, the density decreases.</strong>The growth density of Shewanella is expressed by OD at 600nm and different colors represent different concentrations of Ag+ from 0 μM to 20 μM. The three groups of histograms represent SW-MR-1, SW-MR-Atox1, and SW-MR-Atox1(kanamycin) respectively from left to right.](https://static.igem.wiki/teams/4134/wiki/wiki/results/02-silver-tolerant.png)

## 2. Plasmid Construction and Electrical Transformation

BpfA(C-terminal 1000bp), AgBP2+KanR(910bp), and AggC(N-terminal 1000bp) fragment is amplified from the available plasmid in our laboratory, and the PCR product is purified by agarose gel electrophoresis (figure3).

![Figure3. <strong>PCR product purified by agarose gel electrophoresis. </strong>](https://static.igem.wiki/teams/4134/wiki/wiki/results/03-bpfa-agbp2-kanr-aggc.png)

The existing plasmids are digested by EcoR1 and Kpn1, and the pUC57mini plasmid skeleton is recovered by agarose gel electrophoresis (figure4).

![Figure4. <strong>pUC57mini plasmid skeleton recovered by agarose gel electrophoresis.</strong>](https://static.igem.wiki/teams/4134/wiki/wiki/results/04-to-get-puc57mini.png)

Three target fragments BpfA, AgBP2+KanR, and AggC are sequentially linked in pUC57mini plasmid (figure5) using multi-fragment seamless cloning technology (Novezan ClonExpress®). Then we get the target plasmid.

![Figure5. <strong>Multi-fragment seamless cloning.</strong>](https://static.igem.wiki/teams/4134/wiki/wiki/results/05.png)

Then the constructed plasmid is sequenced to confirm whether it is consistent with the previous design, pUC57mini-BpfA-AgBP2-Linker-KanR-loxP-AggC [[BBa\_K4134078]](http://parts.igem.org/Part%3ABBa_K4134078), which means that the sequence matching degree reaches 100%.

![Figure6. <strong>pUC57mini-BpfA-AgBP2-Linker-KanR-loxP-AggC \[BBa_K4134078\].</strong>](https://static.igem.wiki/teams/4134/wiki/wiki/results/06.png)

The above procedure is repeated by replacing AgBP2 with Atox1, and we get the backup plasmid, pUC57mini-BpfA-Atox1-Linker-KanR-loxP-AggC [[BBa\_K4134067]](http://parts.igem.org/Part%3ABBa_K4134067), which ensures the electrical transformation of Shewanella carried out smoothly.

![Figure7. <strong>pUC57mini-BpfA-Atox1-Linker-KanR-loxP-AggC \[BBa_K4134067\].</strong>](https://static.igem.wiki/teams/4134/wiki/wiki/results/07.png)

First, we introduce the plasmid into E.coli DH 5α for amplification, then we recover it by alkali lyse-adsorption column method, and cleave it by EcoRⅠ and KpnⅠ. Lastly, we purify the target DNA fragments by agarose gel electrophoresis (figure8).

![Figure8. <strong>target DNA fragments purified by agarose gel electrophoresis.</strong>](https://static.igem.wiki/teams/4134/wiki/wiki/results/08-to-get-lineardna.png)

In the first round of experiments, we use AgBP2 as a silver-binding protein to build the device [[BBa\_K4134077]](http://parts.igem.org/Part%3ABBa_K4134077) and screen recombinants for ampicillin resistance. However, the electrotransformation fails many times. In several repetitions, we learn that the electrotransformation of Shewanella is relatively difficult. So, we redesign the device [[BBa\_K4134066]](http://parts.igem.org/Part%3ABBa_K4134066), try another silver-binding protein Atox1, and continuously adjust the voltage intensity of electrotransformation. As a consequence, we finally get a qualified recombinant with kanamycin resistance (KanR) at a lower voltage (1.2kV) of electrical pulses. And AtoxⅠ, the upstream gene of KanR is proved to be displayed on the surface of S. oneidensis.

![Figure9. <strong>The whole process.</strong> First it is our plasmid, pUC57mini-BpfA-Atox1/AgBP2-Linker-KanR-loxP-AggC, then it is introduced into E.coli DH 5α for amplification, recovered, cleaved and purified. Finally, the resulting linear DNA is added into Shewanella.](https://static.igem.wiki/teams/4134/wiki/wiki/results/09.png)

## 3. The Experiments for Biofilm Growth

The modified Shewanella is cultured in LB Culture without Ag+, and the OD at 590nm is measured after 24h and 48h (figure10) to reflect the biofilm thickness of adherent growth Shewanella. The results show that the biofilm thickness of the modified Shewanella was slightly higher than that of the control group.

![Figure10. **The biofilm thickness of the SW-MR-Atox1 is higher than that of the SW-MR-1 in the same condition.** When OD is at 590nm, whether the time is 24h or 48h, the value of the SW-MR-Atox1 is higher than that of the SW-MR-1.](https://static.igem.wiki/teams/4134/wiki/wiki/results/10.png)

## 4. Silver Nanoparticles Detection

1mM \(\ce{AgNO3}\) solution is added to the culture to detect silver nanoparticles:

* SW is the control group, it is wild-type Shewanella.
* SW-Atox1 (SA) is the experimental group, and the silver ion-binding protein Atox1 is fused and expressed in the C-terminal of membrane protein BpfA.

**TEM**

The results of transmission electron microscopy (TEM) (figure11(a))show that silver nanoparticles are indeed obtained on the cell surface, and compared with the wild type, the size of the recombinant nanoparticles was smaller and the density was higher.

![Figure11(a). **Silver nanoparticles under transmission electron microscopy (TEM).**  There are many quantum dots (silver nanoparticles) attached to the SW-MR-1, while SW-MR-Atox1 has more clusters of silver nanoparticles than it.](https://static.igem.wiki/teams/4134/wiki/wiki/results/11.png)

![Figure11(b). **TEM nanoparticle size and elemental analysis**Sw-Atox1 AgNPs are relatively small in size, with most nanoparticles of 10-15nm.](https://static.igem.wiki/teams/4134/wiki/wiki/results/12.png)

**UV-visible Spectroscopy**

The characteristic absorbance of silver nanoparticles (410nm) is detected (figure12). SW-AtoxⅠ has more silver nanoparticles than the control group.

![Figure12(ABC). **The characteristic absorbance of silver nanoparticles.** Silver nanoparticles have their characteristic absorption peak at 410 nm, and it shows that the content of silver nanoparticles in SW-Atox1 reaches the peak in anaerobic conditions.](https://static.igem.wiki/teams/4134/wiki/wiki/results/13-ab-c.jpg)

**XPS (X-Ray Photoelectron Spectroscopy)**

X-Ray Photoelectron Spectroscopy (XPS) can not only determine the surface elements and content but also the valence of elements, indicating that SW-AtoxⅠ has more elemental silver than the control group (figure13).

![Figure13. **The results of XPS.** It shows that elemental silver appears in about 3keV, 22keV, and 25keV, and SW-AtoxⅠ has more elemental silver than SW.](https://static.igem.wiki/teams/4134/wiki/wiki/results/14.png)

**Energy Dispersive X-ray Spectroscopy (EDS)**

Energy Dispersive X-ray Spectroscopy (EDS) is used to detect the composition and content of elements, indicating that SW-AtoxⅠ has higher silver content than the control group (figure14).

![Figure14. **The results of EDS.** The binding energy of elemental silver appears in 367.8eV and 373.8eV. It proves that silver nanoparticles have the same peak as the elemental silver, and SW-AtoxⅠ has more elemental silver than SW.](https://static.igem.wiki/teams/4134/wiki/wiki/results/15.png)

Determination of Silver Nanoparticles under Different Silver Concentrations (figure15).

![Figure15. **(A) Pictures of different strains and Ag+ concentrations after 12h incubation** The first row is the control group (SW) and the second row is the experimental group (SW-Atox1). Silver concentrations are 10μM, 20μM, 100μM and 1000μM respectively from left to right. **(B) Absorption for different strains and Ag+ concentrations, (C) Absorption for different strains, Ag+ concentrations, and incubation time, (D) Absorption for different strains, Ag+ concentrations, and the volume of the reaction** Silver nanoparticles have their characteristic absorption peak at 410 nm, and it shows that the content of silver nanoparticles in SW-Atox1 reaches the peak in anaerobic conditions. ](https://static.igem.wiki/teams/4134/wiki/wiki/results/16.jpg)

## 5.Half-cell Experiment

In the half-cell experiment, we build a three-cell system and designed four working electrodes (figure16).

They are ordinary carbon paper, ordinary carbon paper + reduced graphene oxide, and further, adding chemically synthesized nano-silver particles, and bio-synthesized nano-silver particles in this experiment. The I-t curve was measured at a constant voltage of 0.2V. The electrode with improved stains obtains a higher peak current than the ordinary electrode.

| Stains | Anode material | Maximum current (μA) | Peak arrival time (h) |
| --- | --- | --- | --- |
| SW | Carbon paper | 7.02 | 12 |
| Carbon paper-rGO | 8.9 | 22 |
| Carbon paper-rGO-AgNPs (chemically synthesized) | 17.5 | 62 |
| Carbon paper-rGO-AgNPs (bio-synthesized as we designed) | 6.75 | 114 |
| SW-Atox1 | Carbon paper | 4.7 | 30 |
| Carbon paper-rGO | 8.13 | 20 |
| Carbon paper-rGO-AgNPs (chemically synthesized) | 15.6 | 62 |
| Carbon paper-rGO-AgNPs (bio-synthesized as we designed) | 12.3 | 114 |

![Figure16(d). **Different anode materials are tested.** It shows that only rGO/1mM can increase the efficiency of SW-Atox1, while carbon, rGO, and rGO/AgNPs show opposite results.](https://static.igem.wiki/teams/4134/wiki/wiki/results/17.jpg)

In addition, comparing the chemically synthesized silver nanoparticles with the biosynthetic ones, we use 5% as much silver as is used in our reference to achieve a similar max current intensity (figure17).

![Figure17. **The strain (the electrode without nano-silver) is improved.** The concentration of working fluid \(\ce{AgNO3}\) is 1mM, only 5% of that of the electrode with nano-silver.](https://static.igem.wiki/teams/4134/wiki/wiki/results/18.png)

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