iGEM 2023 — Team cug-china

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List

---

## MFC setup

Bioelectronic device setup and electrochemistry characterization.

Two-chamber MFCs were setup by following the previously established method. The two chambers were separated by proton exchange membranes (Nafion 117 membrane, Shanghai, China). The anodic and cathodic electrodes were carbon cloth (WOS1002, Taiwan Ce Tech) with the size (1 cm × 1 cm, i.e., the geometric area is 1 cm2) and the size (2.5 cm × 3 cm), respectively. The catholyte contains 50 mM K3[Fe(CN)6], 50 mM K2HPO4 and KH2PO4. The anode chamber was filled with 150 mL of M1medium without electron acceptor lactic acid. Cultures of different MR-1 strains were injected into anode chamber at an optical density (OD600) of 0.4 and incubated at 30°C. The anode and cathode were connected by 2 kΩ resistances, and the voltage outputs were recorded in real time. Experiments were conducted with triplicates. To demonstrate the sensitivity of MFCs-based biosensor using different MR-1 biofilms as sensing elements, we added NaAsO₂ and CdCl2 with different final concentrations ranging when voltage outputs peaked and constitutively recorded voltage outputs of MFCs.

## Fe(3)-reduction assay

Fe(III)-reduction assay.

For Fe(III)-reduction assays, the MR-1 strains were pre-cultured in the M1 medium with fumarate as an electrons acceptor and H2 as an electrons donor . Reduction of 20 mM of ferrihydrite was conducted at 30°C with MR-1 cells at starting OD600 of 0.1. All procedures were performed in an anaerobic chamber (Coy Laboratory Products Inc., Grass Lake, MI, USA) that was filled with 10% H2, 20% CO2 and 70% N2. The reduced Fe(2) was measured with a ferrozine assay (Stookey 1970).

## PCR Purification

Material

Ethanol 96-100%

Isopropanol

3M sodium acetate, pH 5.2 (may be necessary)

Microcentrifuge

1.5 or 2 mL microcentrifuge tubes

Heating block or water bath

GeneJET PCR Purification Kit (produced by Thermo Fisher Scientific®)

Before Starting

Examine the Binding Buffer for precipitates before each use. Re-dissolve any precipitate by warming the solution to 37 ℃ and cooling to 25 ℃ .

Protocol

1.Add a 1:1 volume of Binding Buffer to completed PCR mixture (e.g. for every 100 µL of reaction mixture, add 100 µL of Binding Buffer). Mix thoroughly. Check the color of the solution. A yellow color indicates an optimal pH for DNA binding. If the color of the solution is orange or violet, add 10 µL of 3 M sodium acetate, pH 5.2 solution and mix. The color of the mix will become yellow.

2.Optional: if the DNA fragment is ≤ 500 bp, add a 1:2 volume of 100% isopropanol (e.g., 100 µL of isopropanol should be added to 100 µL of PCR mixture combined with 100 µL of Binding Buffer). Mix thoroughly.

Note:If PCR mixture contains primer-dimers, purification without isopropanol is recommended. However, the yield of the target DNA fragment will be lower.

3.Transfer up to 800 µL of the solution from step 1 (or optional step 2) to the GeneJET purification column. Centrifuge for 30-60 s. Discard the flow-through.

Notes: If the total volume exceeds 800 µL, the solution can be added to the column in stages. After the addition of 800 µL of solution, centrifuge the column for 30-60 s and discard flowthrough. Repeat until the entire solution has been added to the column membrane. Close the bag with GeneJET Purification Columns tightly after each use!

4.Add 700 µL of Wash Buffer (diluted with the ethanol as described on p. 3) to the GeneJET purification column. Centrifuge for 30-60 s. Discard the flow-through and place the purification column back into the collection tube.

5.Centrifuge the empty GeneJET purification column for an additional 1 min to completely remove any residual wash buffer.

Note.:This step is essential as the presence of residual ethanol in the DNA sample may inhibit subsequent reactions. 6

6.Transfer the GeneJET purification column to a clean 1.5 mL microcentrifuge tube (not included). Add 50 µL of Elution Buffer to the center of the GeneJET purification column membrane and centrifuge for 1 min.

Note:For low DNA amounts the elution volumes can be reduced to increase DNA concentration. An elution volume between 20-50 µL does not significantly reduce the DNA yield. However, elution volumes less than 10 µL are not recommended.

If DNA fragment is >10 kb, prewarm Elution Buffer to 65℃ before applying to column.

If the elution volume is 10 µL and DNA amount is ≥5 µg, incubate column for 1 min at room temperature before centrifugation.

7.Discard the GeneJET purification column and store the purified DNA at -20℃.

## Glycerol preservation

Material

high pressure steam sterilizer

glycerin

Protocol

1.A 40% glycerol solution was prepared in a volume ratio of 2:3 for strain preservation.

2.After the preparation is completed, sterilize with high pressure steam at 121 °C for 20 min, and store at room temperature.

3.When using, the bacteria should be preserved according to the volume ratio of glycerol and bacterial liquid to 1:1.

## DNA fragment extraction from agarose gels

Material

Ethanol 96-100%

Isopropanol

3M sodium acetate, pH 5.2 (may be necessary)

Microcentrifuge

1.5 or 2 mL microcentrifuge tubes

Heating block or water bath

GeneJET Gel Extraction Kit (produced by Thermo Fisher Scientific®)

Before Starting

Examine the Binding Buffer for precipitates before each use. Re-dissolve any precipitate by warming the solution to 37 ℃ and cooling to 25 ℃ .

Protocol

1.Excise gel slice containing the DNA fragment using a clean scalpel or razor blade. Cut as close to the DNA as possible to minimize the gel volume. Place the gel slice into a pre-weighed 1.5 mL tube and weigh. Record the weight of the gel slice.

Note:If the purified fragment will be used for cloning reactions, avoid damaging the DNA through UV light exposure. Minimize UV exposure to a few seconds or keep the gel slice on a glass or plastic plate during UV illumination.

Add 1:1 volume of Binding Buffer to the gel slice (volume: weight) (e.g., add 100 µL of Binding Buffer for every 100 mg of agarose gel).

Note: For gels with an agarose content greater than 2%, add 2:1 volumes of Binding Buffer to the gel slice.

3.Incubate the gel mixture at 50-60 ℃ for 10 min or until the gel slice is completely dissolved. Mix the tube by inversion every few minutes to facilitate the melting process. Ensure that the gel is completely dissolved. Vortex the gel mixture briefly before loading on the column. Check the color of the solution. A yellow color indicates an optimal pH for DNA binding. If the color of the solution is orange or violet, add 10 µL of 3 M sodium acetate, pH 5.2 solution and mix. The color of the mix will become yellow.

4.Optional: use this step only when DNA fragment is ≤500 bp or >10 kb long.

If the DNA fragment is ≤500 bp, add 1 gel volume of 100% isopropanol to the solubilized gel solution (e.g. 100 µL of isopropanol should be added to 100 mg gel slice solubilized in 100 µL of Binding Buffer). Mix thoroughly.

If the DNA fragment is >10 kb, add 1 gel volume of water to the solubilized gel solution (e.g. 100 µL of water should be added to 100 mg gel slice solubilized in 100 µL of Binding Buffer). Mix thoroughly.

Transfer up to 800 µL of the solubilized gel solution (from step 3 or 4) to the GeneJET purification column. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.

Note: If the total volume exceeds 800 µL, the solution can be added to the column in stages. After each application, centrifuge the column for 30-60 s and discard the flow-through after each spin. Repeat until the entire volume has been applied to the column membrane. Do not exceed 1 g of total agarose gel per column. In addition ,Close the bag with GeneJET Purification Columns tightly after each use!

6.Optional: use this additional binding step only if the purified DNA will be used for sequencing. Add 100 µL of Binding Buffer to the GeneJET purification column. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.

7.Add 700 µL of Wash Buffer (diluted with ethanol as described on p. 3) to the GeneJET purification column. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.

8.Centrifuge the empty GeneJET purification column for an additional 1 min to completely remove residual wash buffer.

Note:This step is essential to avoid residual ethanol in the purified DNA solution. The presence of ethanol in the DNA sample may inhibit downstream enzymatic reactions.

Transfer the GeneJET purification column into a clean 1.5 mL microcentrifuge tube (not included). Add 50 µL of Elution Buffer to the center of the purification column membrane. Centrifuge for 1 min.

Note:For low DNA amounts the elution volumes can be reduced to increase DNA concentration. An elution volume between 20-50 µL does not significantly reduce the DNA yield. However, elution volumes less than 10 µL are not recommended.

If DNA fragment is >10 kb, prewarm Elution Buffer to 65 °C before applying to column.

If the elution volume is 10 µL and DNA amount is ≤ 5 µg, incubate column for 1 min at room temperature before centrifugation.

10.Discard the GeneJET purification column and store the purified DNA at -20 °C

## Make competent cell

Material

LB plates

Note: In the experiment, different engineering bacteria were added with corresponding antibiotics

1.5 mL centrifuge tube

centrifuge

Solution A

Solution B

Protocol

1.Take out the preserved strain from the -80℃ ultra-low temperature refrigerator, streak it on the LB plate, and cultivate it overnight at 37℃ to pick out a single colony to LB liquid medium for expansion.

2.Take 20 mL of logarithmic bacterial liquid with an OD600 value between 0.35 and 0.5, aliquot into a 1.5 mL centrifuge tube, and place it on ice for 10 minutes to stop culturing.

3.Use a centrifuge at 4℃ and 5000 rpm for 5 minutes to collect bacteria and discard the supernatant. Add 100 μL of low-temperature Solution A to the centrifuge tube, and tap the centrifuge tube to suspend the bacterial solution.

4.Centrifuge again at 4℃, 5000 rpm for 5 minutes to collect the cells, then add 100 μL of pre-cooled Solution B to the centrifuge tube, and flick to suspend the cells.

5.Immediately store the heat-shock competent cells prepared above at -80℃ for use in the next step.

## Electrocompetent Cell Transformation

Material

Electrocompetent cells

Assembly product

LB plates

Note: In the experiment, different engineering bacteria were added with corresponding antibiotics.

Electroporation cuvette

Electroporation machine

Pipette with tips

Incubator cabinet

Protocol

1.Take glycerol strains to LB plate, pick out monoclonal colony and expand the culture overnight on a shaker at 30℃ and 180 rpm.

Note: *S.oneidensis* was cultured at 30℃, *E. coli* at 37℃.

2.Take 1 mL of bacterial solution into a centrifuge tube, centrifuge at 15,000 rpm for 2 min to collect cells, and discard the supernatant.

3.Use 1 mL of 0.3 mol/L sterilized sucrose solution to resuspend the cells by pipetting up and down, centrifuge again at 15,000 rpm for 2 min, and discard the supernatant.

4.Repeat step 3.

5.Add 100 μL of sucrose solution again to resuspend, add about 100 ng of recombinant plasmid to the competent cells, and mix well. The mixture was added to the electro-rotating cup, electric shock was performed at 2.5 kV for 5 ms, and 900 μL of LB liquid medium was quickly added to resuspend the bacteria.Then placed on a shaker at 200 rpm and activated at 30 ℃ for 2 h.

6.Centrifuge at 5000 rpm for 5 min and discard the supernatant. Use 100 μL of LB liquid medium, pipette to resuspend, spread on LB plate by dilution plate coating method, and incubate upside down in a 30℃ bacterial incubator for 12-20 h until colonies appear.

7.Pick monoclonal colonies for PCR verification, screen out strains with correct bands for sequencing, and strains with correct sequencing for glycerol storage.

Note：Antibiotics can be determined according to the plasmid.

## Transformation

Material

LB plates

Note: In the experiment, different engineering bacteria were added with corresponding antibiotics.

1.5 mL centrifuge tube

competent cells

recombinant plasmid

ice box

incubator

Protocol

1.Take out the frozen competent cells from the -80℃ ultra-low temperature freezer and thaw on ice;

2.Take 10 μL of the recombinant plasmid and add it to 100℃ of heat-shock competent cells, mix them together carefully, and place in an ice box for about 30 minutes;

3.Heat in a water bath at 42℃, heat shock for 90 s, and then immediately cool on ice for 2-3 minutes;

4.Add 900 μL of LB liquid medium prepared in advance to the centrifuge tube, and then place the bacterial solution at 150 rpm, Incubate at 37℃ for 1 h.

5.The LB liquid culture was preheated in a 37℃ incubator, and the activated bacterial solution was centrifuged at 5000 rpm.

6.Discard 900 μL of the supernatant, resuspend with the remaining 100 μL, spread the bacterial solution onto LB plates using the dilution plate coating method, and invert overnight in a 37℃ incubator.

7.Pick monoclonal colonies for PCR verification, screen out strains with correct bands for sequencing, and strains with correct sequencing for glycerol storage.

## LB Liquid Culture Medium

Material

Materials for 1 L:

Yeast Extract: 5 g

Tryptone: 10 g

NaCl: 10 g

Protocol

1.Preparation of LB Liquid Culture Medium

2.Measure 10 g of Tryptone, 5 g of Yeast extract, and 10 g of NaCl. Place them in a 1,000 mL sterilized bottle.

3.Add 0.5 L of deionized water, shake the bottle until the solids completely dissolve. Then, add more deionized water until the solution has a volume of 1 L.

4.Place the bottles into the autoclave for sterilization, under 121°C for 30 minutes.

5.Let the bottles cool down under room temperature. The solution would preserve under room temperature for a maximum of 7 days.

6.15 g/L agarose can be added to form a solid medium, and after sterilization, it can be divided into solid agar plates

## Colony PCR Protocol

Material

Takara PrimeSTAR® Max DNA Polymerase

Colony template

Primers

Nuclease-free 0.2 mL PCR tubes

PCR instrument

Protocol

1.The choice of reaction system

| Components | Volume |
| --- | --- |
| DNA template Few colony | |
| 10 μM Forward Primer 1 μL | |
| 10 μM Reverse Primer 1 μL | |
| PrimeSTAR® Max DNA Polymerase 12.5 μL | |
| Sterile deionized water 9.5 μL | |

2.The choice of PCR program:

| Step | Temperature | Duration | Loops |
| --- | --- | --- | --- |
| Preheat 98 ℃ | - | - | |
| 1 98 ℃ | 5 min | 30 loops | |
| 2 translateX | 1 min | 30 loops | |
| 3 72 ℃ | 30 s/kb | 30 loops | |
| 4 72 ℃ | 5 min | - | |
| 5 15 ℃ | Hold | - | |

*Note: x is the annealing temperature of the reaction, usually 2℃ to 3℃ lower than Tm of the primer.*

The calculation of Tm: Tm = 4(G+C) + 2(A+T)

## Fluorescence Intensity Measurement

Material

LB medium

Note: In the experiment, different engineering bacteria were added with corresponding antibiotics.

Colony template

PBS buffer

96-well plate

Micro plate reader (produced by gene5®)

Protocol

1.Pick a single colony from the plate, inoculate it into 3-5mL LB liquid medium, and inoculate it at 30℃ for about 12 h with shaking. Incubate overnight.

2.The next day, inoculate 50 μL of the overnight culture into 5 mL of fresh LB medium. After culturing to 6 h, 12 h and 24 h, take 200 μL of bacterial liquid from each sample.

Note: If necessary, add different concentrations of IPTG (0, 10-5, 10-4, 10-3, 10-2, 10-1 mM).

3.Centrifuge at 4000 rpm for 10 min and discard the supernatant. Resuspend in 1 mL of PBS, centrifuge at 4000 rpm for 10 min, and discard the supernatant. Resuspend in 1 mL of PBS, centrifuge at 4000 rpm for 10 min, and discard the supernatant. Resuspend in 200 μL PBS, and measure the fluorescence intensity in a 96-well plate.

Micro plate reader parameters are shown below.

| Mode | Fluorescence Bottom Reading |
| --- | --- |
| Excitation Wavelength 485 nm | |
| Emission Wavelength 535 nm | |
| Excitation Bandwidth 9 nm | |
| Emission Bandwidth 20 nm | |
| Gain 60 Manual | |
| Number of Flashes 25 | |
| Integration Time 20 μs | |
| Lag Time 0 μs | |
| Settle Time 0 ms | |
| Part of Plate A1-A12 | |

Table 1 Micro Plate Reader Parameter

| Ingredient | dosage |
| --- | --- |
| NaCl 8.000 g | |
| KCl 0.200 g | |
| KH2PO4 0.240g | |
| Na2HPO4 3.640g | |

## DNA Gel Electrophoresis Protocol

Material

50 × TAE concentrate Solution

Agarose (produced by Biowest®) —DNA dye (TransGen® GelStain)

100mL flask

Distilled water

Microwave oven

DNA samples

10 ×Loading buffer (produced by New England Biolabs ®)

DNA marker (produced by TsingkeBiotechnologyCo.,Ltd.®)

Electrophoresis instrument (produced by Bio-Rad®)

Before Starting

Dilute 50 ×TAE concentrate Solution to 1 ×TAE buffer with distilled water.

Add 10 ×loading buffer into marker and DNA samples. Loading buffer should occupy 10% of total volume.

Note:If the Taq enzyme used in PCR already has a premixed DNA dye, there is no need to add loading buffer

Protocol

1.Weigh 0.3 g agarose in a flask.

2.Add 30 mL 1 × TAE buffer into the flask from Step 1.

3.Melt the agarose in the microwave oven (medium-high heat, about 3 minutes).

Note: In order to make the gel more even, reheating the agarose is recommended.

4.Add 3 uL TransGen® GelStain, mix by shocking.

5.Insert slots to assemble gel pouring apparatus through the gate.

6.Pour agarose gel into the gel tray.

7.Cool for 40 minutes to FULLY solidify the DNA agarose gel.

8.Remove the pouring apparatus, place the gel into an electrophoresis instrument.

9.Pipet marker and DNA samples which have been mixed with loading buffer into the slots.

10.Turn on the electrophoresis instrument, set the working electric current at 120-150 mA and the working voltage at 120 V.

11.Electrophoresis for 20-35 minutes.

Note: When the blue strips move to two-thirds of the gel, the electrophoresis could be stopped.

12.Turn off the instrument and place the gel in the gel formatter to take and save the photo.

Table1 TAE running buffer (50x)

| Ingredient | Dosage |
| --- | --- |
| Tris Base 242 g/L | |
| Ethylenediaminetetraacetic acid 37.2 g/L | |
| Acetic acid 57.1 mL/L | |
| ddH2O 700 mL | |
| Adjust the pH of the system to 8.3 with 1 mol/L sodium hydroxide solution, and then make up to 1 L with ddH2O |

## PCR(Polymerase Chain Reaction) Protocol

Material

Cwbio 2x Es Taq MasterMix(Dye)

Template DNA

ddH2O

Primers

Nuclease-free PCR tubes

PCR imstrument

Protocol

1.Set up a reaction system:

| Components | Volume |
| --- | --- |
| DNA template 1 L(<200 ng) | |
| 10 uM Forward Primer 1 μL | |
| 10 μM Reverse Primer 1 μL | |
| 2x Es Taq MasterMix(Dye) 25 μL< | |
| ddH2O 22 μL | |

2.Choose the PCR program based on the reaction:

| Step | Temperature | Duration | Loops |
| --- | --- | --- | --- |
| 1 95 ℃ | 4 min | - | |
| 2 98 ℃ | 10 sec | 35 loops | |
| 3 Tm | 5 sec | 35 loops | |
| 4 72 ℃ | 5 sec/kb | 35 loops | |
| 5 72 ℃ | 10 min | - | |
| 6 12 ℃ | Hold | - | |

*Note: If the template is from a colony, add 1 μL ddH2O and add a few templates colony using pipette tips.*

## Plasmid Extraction Protocol

Material

AG SteadyPure Plasmid DNA Extraction Kit

Vortex

Microcentrifuge

Nuclease-free 1.5 mL microcentrifuge tubes

Sterile deionized water

100% ethanol

Before Starting

Add the RNase A Solution included in the kit to the Resuspension Solution and mix throughly. After the additon of RNase A, the Resuspension Solution should be stored at 4 ℃.

Add the indicated volume of ethanol (96-100%) to Wash Solution II (concentrated) prior to first use.

Make sure to wear gloves.

Protocol

1.Add a 1.5 mL of bacterial culture to microcentrifuge tubes and harvest the cells by centrifugation for 10 min at 5,000 x g. Discard the supernatant.

2.Resuspend the pelleted cells in 250 uL of the Resuspension Solution. The bacteria should be resuspended completely by vortexing and no cell clumps remain.

3.Add 250 uL of the Lysis Solution and mix throughly by inverting the tube 4-6 times until the solution becomes viscous and slightly clear.

Note:Do not vortex to avoid shearing of chromosomal DNA. Do not incubate for more than 5 min to avoid denaturation of supercoiled plasmid DNA.

4.Add 350 uL of the Neutralization Solution and mix immediately and throughly by inverting the tube 4-6 times. The neutralized bacterial lysate should become cloudy.

5.Centrifuge at 12,000 x g for 5 min to pellet cell debris and chromosomal DNA.

6.Transfer the supernatant to the supplied GeneJET spin column bt decanting or pipetting. Avoid disturbing or transferring the white precipitate.

7.Centrifuge at 12,000 x g for 1 min. Discard the flow-through and place the column back into the same collection tube.

8.Add 500 uL of the Wash Solution to the spin column. Centrifuge at 12,000 x g for 1 min and discard the flow-through. Then place the column back into the same collection tube.

9.Repeat the step 8.

10.Centrifuge the empty tube for an additional 1 min to remove residual Wash Solution.

11.Transfer the spin column into a fresh 1.5 mL microcentrifuge tube. Add 50 uL of the sterile deionized water to the center of spin column membrane to elute the plasmid DNA. Incubate for 2 min at room temperature.

12.Discard the column and store the purified plasmid DNA at -20℃.

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## Index

Abstract
Phase 1
Phase 2
Phase 3

---

## Abstract

Bioelectrochemical sensors, in which electrochemically active biofilms (EABs) are employed as bio-elements, sense analytes of interest by converting metabolic changes to easily detectable electrical signals. EABs are formed by electroactive bacteria capable of exchanging electrons with electrodes. The self-immobilization, self-sustainability, and high robustness of EABs make EAB-enabled biosensors show promise in environmental applications, such as water quality monitoring. This year, our team utilized the electroactive microorganism *Shewanella oneidensis* MR-1 as the chassis cell to construct an EAB-based biosensor for sensitive and specific detection of arsenic.

Firstly, we engineered the biofilm of S. *oneidensis* MR-1 by knocking out its *ptpA* gene that is associated with the biosynthesis of extracellular polysaccharides, resulting in a more conductive *S. oneidensis* MR-1 biofilm formed on the anode of a microbial fuel cells (MFCs). The enhanced conductivity of *S. oneidensis* MR-1 biofilm improved the sensitivity of EAB-based biosensor for arsenic detection. However, the biosensor exhibited a low specificity for arsenic detection.

To improve the specificity of our biosensor, we selected the P*ars*-*arsR* system from *Escherichia coli* as the arsenic sensing components. This arsenic responsive transcription system was used to control the expression of cytochrome genes *cymA* or *mtrC* that is involved in extracellular electron transfer in *S. oneidensis* MR-1. By transferring this gene circuit into double deletion strains Δ*ptpAmtrC* or Δ*ptpAcymA*, the presence of arsenic can be converted to increased electrical signals, as arsenic can promote the expression of *cymA* or *mtrC* to recover the ability of EET in *S. oneidensis* MR-1.

To further optimize the biosensor, we construct a self-amplifier system that does not rely on exogenous inducers (see Part improve for details) by modifing the LuxR protein from *Vibrio fischeri*. We combined this self-amplifier system with the arsenic sensing circuit to enhance the sensitivity and specificity of the biosensor.

Taken together, this work includes three phases: engineering of *S. oneidensis* electroactive biofilm, construction and validation of the arsenic sensing system, and construction of the self-amplifier system.

## Phase 1: Engineering of *S. oneidensis* Electroactive Biofilm

The electroactive biofilm is composed of electroactive cells and the extracellular polymeric substances (EPS) they secrete. EPS mainly consists of extracellular polysaccharides, proteins, DNA, and electroactive compounds such as cytochromes and nanowires. Among these components, extracellular polysaccharides is unconductive, resulting in inefficient EET. The *ptpA* gene plays a key role in the synthesis of extracellular polysaccharides. Thus, the deletion of *ptpA* can reduce the amount of extracellular polysaccharides in biofilm matrix to enhance the conductivity of the *S. oneidensis* MR-1 biofilm. It has been reported that the enhanced EET efficiency of EAB can improve the sensitivity of EAB-based biosensors. Thus, we knocked out the *ptpA* gene from the *S. oneidensis* MR-1 genome and applied Δ*ptpA* in MFCs. The results showed that Δ*ptpA* produced higher electrical generation than the WT, and Δ*ptpA* biofilm exhibited higher conductivity. To test if this conductive biofilm can be used as sensitive elements to improve the sensitivity of sensor, we comparatively analyzed concentration-response curves of MFCs inoculated with *S. oneidensis* MR-1 wild type and Δ*ptpA* via As3+ shocks. The results show that the EAB-based sensors with Δ*ptpA* biofilms can detect lower concentrations of As3+ than ones with wild type biofilms.

However, other heavy metal, such as cadmium, also can change electrical signal of sensor, indicating low specificity of this *S. oneidensis* biofilm-based biosensor.

![Pulpit rock](https://static.igem.wiki/teams/4767/wiki/project-engineering/en-1.png)

Fig 1. *ptpA* gene knockout verification

## Phase 2: Construction and Validation of the Arsenic Sensing System

To improve the specificity of sensor, we cloned an arsenic responsive transcription system, i.e., the P*ars*-*arsR* system to control the expression of EET-associated genes. The arsenic-inducible promoter (P*ars*) is negatively regulated by ArsR. When arsenic is excluded from a cell with these genetic components, the binding kinetics between ArsR and its associated operator region within P*ars* are relatively strong, blocking the transcription of downstream genes. The dynamics of the circuit change, however, when arsenic enters the cell. In this condition, arsenic associates with ArsR, inducing a conformational change that leads to its dissociation from the P*ars* operator. Because the binding kinetics between ArsR and the ABS become more unfavorable with increasing arsenic concentrations, GFP levels increase with increasing arsenic presence. To verify that the P*ars*-*arsR* system from *E.coli* can work properly in *Shewanella* cells, we firstly fused *arsR* with *gfp* to obtain [Part 1](http://parts.igem.org/Part%3ABBa_K4767000) (Fig.2). Theoretically, when arsenic concentration increases in the system, fluorescence intensity increases (see results).

![Pulpit rock](https://static.igem.wiki/teams/4767/wiki/project-engineering/en-2.png)

Fig 2. Structure of part 1 and Colony RPC result of part 1

The *cymA* and *mtrC* genes encode c-type cytochromes that play important roles in EET of *S. oneidensis* MR-1. Previous studies showed that the deletions of *cymA* or *mtrC* disrupted the ability of *S. oneidensis* MR-1 to generate electricity in MFCs. We knock out the *cymA* and *mtrC* genes from Δ*ptpA* strain, obtaining the double knockout strain Δ*ptpA**mtrC* or Δ*ptpA**cymA*. Meanwhile, we fused *cymA* and *mtrC* with the P*ars*-*arsR* system, respectively, obtaining [Part 3](http://parts.igem.org/Part%3ABBa_K4767002) and [Part 4](http://parts.igem.org/Part%3ABBa_K4767003) (Fig.3). We transferred Part 3 plasmid and Part 4 plasmid into Δ*ptpA**mtrC* or Δ*ptpA**cymA*. By conducting MFCs experiments, we found that when the cultures of Δ*ptpA**cymA* with Part 3 and Δ*ptpA**mtrC* with [Part 4](http://parts.igem.org/Part%3ABBa_K4767003) were induced by arsenic with different concentrations, the electricity signals enhanced with the increase in arsenic concentrations. However, the cadmium cannot change electricity signals. It indicates that this biosensor can detect specifically detect arsenic (see the result).

![Pulpit rock](https://static.igem.wiki/teams/4767/wiki/project-engineering/en-3.png)

Fig 3. Structure of part 3/4 and Colony RPC result of part 3/4

## Phase 3: Building Amplifiers to Boost Biosensor Performance

To further improve the sensitivity of our biosensor, we constructed the P*luxI*-*luxR* (Δ2-162) positive feedback system (See Project Design) and combined it with the arsenic-responsive transcription system, which were conducted in the cycle 2. Firstly, we fused *luxR* (Δ2-162) with *gfp* to get [Part2](http://parts.igem.org/Part%3ABBa_K4767001) (Fig. 4) and compared it with the original *luxR*. It was verified that it could drive downstream gene expression without the addition of the exogenous AHL in *S. oneidensis* MR-1 (See improved parts).

![auto](https://static.igem.wiki/teams/4767/wiki/project-engineering/en-4.png)

Fig 4. Structure of part 2 and Colony RPC result of part 2

Then, we constructed [Part5](http://parts.igem.org/Part%3ABBa_K4767004)/[6](http://parts.igem.org/Part%3ABBa_K4767005)/[7](http://parts.igem.org/Part%3ABBa_K4767006) and fused *cymA* and *mtrC* with *luxR* (Δ2-162) to obtain the corresponding gene circuis Part 6,7 (Fig5). In the part 5, we used *luxR* (Δ2-162) as the reporter gene in response to the arsenic. With the arsenic level rises, part 5 will produce more LuxR (Δ2-162) as a result. Then the LuxR (Δ2-162) functions as an activator to start the positive feedback loops of part 6/7. LuxR (Δ2-162) can active the gene transcription driven by the *lux* promoter in part 6/7, producing MtrC/CymA and more LuxR (Δ2-162). In this case, LuxR (Δ2-162) works as both reporter and activator, creating a positive feedback loop gene circuit.

![auto](https://static.igem.wiki/teams/4767/wiki/project-engineering/en-5.png)

Fig 5. Structure of part 5,6,7 and Colony RPC result of part 5,6,7

Finally, we insert Part 6/7 into the back of Part 5 to obtain the final *cymA*-enchanced biosensor [Part 8](http://parts.igem.org/Part%3ABBa_K4767007) and *mtrC*-enhanced biosensor [Part 9](http://parts.igem.org/Part%3ABBa_K4767008) (Fig 6). After the modification of the *luxR* (Δ2-162), we applied the amplifier into the arsenic-response system we build. The transcriptional activator, *luxR* (Δ2-162), was used to replace *mtrC* as reporter, and it was regulated by the *arsR*-P*ars* circuit, while *mtrC/cymA* together with a second *luxR* (Δ2-162) was placed under the promoter P*luxI*, which was activated by *luxR* (Δ2-162). When arsenic is present, it activates the expression of *luxR* (Δ2-162) in the first circuit, which turns on the expression of *mtrC/cymA* and *luxR* (Δ2-162) (Part 5)from the following circuit. The second *luxR* (Δ2-162) activates its own expression as well as that of *mtrC/cymA* and forms a positive feedback loop to enhance the output signal from *mtrC/cymA*. These two parts work together as the arsenic EAS-based sensor with the positive feedback amplifier (Part 6 & 7). Our results suggested that, compared with the sensor without positive feedback, the one with a positive feedback amplifier functions well in enhancing the iron reduction rates and better performance in MFC, increasing the detection range, and improving sensitivity.

![Pulpit rock](https://static.igem.wiki/teams/4767/wiki/project-engineering/en-6.png)

Fig 6. Structure of part 8/9 and Colony RPC result of part 8/9

| Strain | usage & detail |
| --- | --- |
| *Shewanella oneidensis*  | |
| WT Wild Type strain | |
| Δ*ptpA* *ptpA* gene mutant strain | |
| Δ*ptpAmtrC* *ptpA/mtrC* gene mutant strain | |
| Δ*ptpAcymA* *ptpA/cymA* gene mutant strain | |
| Part1 Wild Type strain containing the As sensor plasmid Part 1 using *gfp* as reporter gene | |
| Δ*ptpAmtrC*/MtrC (Part 4) *ptpA/mtrC* gene mutant strain contaning As sensor Part 4 using *mtrC* as reporter gene | |
| Δ*ptpAcymA*/CymA (Part 3) *ptpA/cymA* gene mutant strain contaning As sensor Part 3 using *cymA* as reporter gene | |
| Part 2 Wild Type strain containing the modified LuxR-based amplifier Part 2 using *gfp* as reporter gene | |
| P*bad*-*luxI*-P*tac*-*luxR*-P*luxI*-*gfp* Expression of LuxI for AHL production and LuxR are controlled by arabinose-inducible promoter P*bad* and IPTG-inducible promoter P*tac*, respectively | |
| Δ*ptpAmtrC*/MtrC(+)(Part9) *ptpA/mtrC* gene mutant strain contaning As sensor amplifier Part 9 using *mtrC* as reporter gene | |
| Δ*ptpAcymA*/CymA(+)(Part8) *ptpA/mtrC* gene mutant strain contaning As sensor amplifier Part 8 using *cymA* as reporter gene | |

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## Index

Part Ⅰ
Part Ⅱ
Part Ⅲ

---

## 1. Δ*ptpA* Improved the Conductivity of *Shewanella oneidensis* MR-1 Biofilm and Enhanced Sensitivity of *S. oneidensis* MR-1 Biofilm-based Biosensor

Due to its un-conductivity, the polysaccharide in *S. oneidensis* MR-1 biofilm matrix reduced the extracellular electron transfer (EET) in *S. oneidensis* MR-1 biofilm. Thus, we deleted polysaccharide biosynthesis-associated gene *ptpA* from *S. oneidensis* MR-1 genome. Then, the conductivity of *ptpA* gene knockout strain Δ*ptpA* and wild-type (WT) biofilms grown on anodes in microbial fuel cells (MFCs) were investigated. The Δ*ptpA* produced a significantly higher maximum voltage output (153.39 ± 10.27
mV) than the WT biofilm whose peak value was 116.85 ± 8.75 mV (Figure 1A). The performance of MFCs with both strains was further evaluated through the measurement of the polarization curves and the power density curves. As shown in Figure 2B, the slopes of the polarization curve of the MFCs with Δ*ptpA* was much smaller than that with the WT strain (Figure 2B); the maximum power density (MPD) was also improved from 45.96 mW m-2 (WT) to 74.85 mW m-2 (Δ*ptpA*) (Figure 2C). Collectively, the results suggest that the deletion of Δ*ptpA* enhances EET in electrode-attached *S. oneidensis* biofilms.

![Pulpit rock](https://static.igem.wiki/teams/4767/wiki/project-results2/res-1.png)

Figure 1. (A) Voltage output curves, (B) polarization and (C) power density curves of MFCs with the *S. oneidensis* WT and Δ*ptpA* strain.

To test if this conductive biofilm can be used as sensitive elements to improve the sensitivity of electroactive biofilm (EAB)-based sensor, we comparatively analyzed concentration-response curves of MFCs inoculated with Δ*ptpA* and WT via As3+ shocks. EAB-based sensor senses analysts by converting metabolic changes to easily detectable electrical signals. Thus, we firstly tested the inhibitory effect of arsenic toxicity on bacterial growth. WT and Δ*ptpA* were cultured in arsenic-containing medium (sodium arsenite). It was found that a concentration of 100 mg/L of arsenic significantly inhibited the growth of *S. oneidensis* MR-1 (Figure 2), so this concentration was subsequently diluted for toxicity sensor testing in MFC systems.

![Pulpit rock](https://static.igem.wiki/teams/4767/wiki/project-results2/res-2.png)

Figure 2. The growth curves of *S. oneidensis* WT and Δ*ptpA* strain in the medium containing (A) 50 mg/L, (B) 100 mg/L and (C) 500 mg/L As3+.

After MFCs generated stable voltages, As3+ with different concentrations ranging from 0 to 100 mg L-1 was fed into the systems. The voltage outputs of MFCs after As3+ shocks were shown in Figure 3. As3+ shocks contributed to the sharply decreased voltages for both Δ*ptpA* and WT MFCs in 15 mins when As3+ concentrations were above 50 mg/L (Figure 3A and B), whereas only Δ*ptpA* MFCs showed a significant decline in voltage outputs when As3+ concentration was lowed to 25 mg/L (Figure 3C) . These indicate that the EAB-based sensors with Δ*ptpA* biofilms can detect lower concentrations of As3+ than ones with WT biofilms. However, the presence of other heavy metals, such as Cd2+ (10 mg/L) tested in our project, also resulted in a decline in voltage (Figure 3D). It is indictive of un-specificity of our biosensor.

![Pulpit rock](https://static.igem.wiki/teams/4767/wiki/project-results2/res-3.png)

Figure 3. The decreased voltages of MFCs with *S. oneidensis* WT and Δ*ptpA* biofilms under exposure to As3+ with the concentrations including 100 (A), 50 (B), 25 (C) mg L-1 and Cd2+ (D) with the concentration of 10 mg L-1.

## 2. Construction of Arsenic-specific Biosensor

To construct an EAB-based biosensor for specific detection of As3+, we introduced an arsenic responsive transcription system composed of the transcription regulator *arsR* and its corresponding promoter P*ars*. To test whether the arsenic responsive transcription system can work in *S. oneidensis* MR-1, we constructed Part 1 (Figure 4A) in which the green fluorescent protein (*gfp*) as a reporter gene was placed at the downstream of the arsenic responsive transcription system. The MR-1/Part 1, in which *S. oneidensis* MR-1 WT contains the gene circuit Part 1, produced fluorescence signals in the presence of As3+ (Figure 4B and C). The fluorescence increased with an increase in As3+ concentrations (Figure 4B), whereas the increased fluorescence was not observed when Cd2+ was present in the system (Figure 4C). Collectively, the results indicate the feasibility of using *arsR*- P*ars* system to construct a *S. oneidensis* MR-1 biosensor for specific detection of As3+.

![Pulpit rock](https://static.igem.wiki/teams/4767/wiki/project-results2/res-4.png)

Figure 4. (A) Gene circuit contains an arsenic responsive transcription *arsR*- P*ars* system and a reporter gene *gfp*. (B) Dose-response fluorescence curve of an arsenic responsive transcription system to different concentrations of As3+. (C) The response of arsenic responsive transcription system to As3+ and Cd2+. (NS: No significance. \* 0.01< P < 0.05.\*\* P < 0.01)

Previous studies showed that the deletion of MtrC or CymA, which are important cytochromes for extracellular electron transfer (EET) pathway in *S. oneidensis* MR-1, significantly disrupted the ability of *S. oneidensis* MR-1 to reduce Fe(Ⅲ) or anode in MFCs. To convert the presence of As3+ to the electrical signals, we used the arsenic responsive transcription system to control the expression of MtrC or CymA. As shown in Figure 5A, we constructed gene circuits Part 3 and Part 4. Then, Part 3 and Part 4 were transformed into Δ*cymA/ptpA* (double deletion strain) and Δ*mtrC/ptpA* (*mtrC* and *ptpA* double deletion strain) , respectively. The resulting strains were named as Δ*cymAptpA* /CymA and Δ*mtrCptpA*/MtrC. In these two strains, the presence of arsenic can induce the expression of MtrC or CymA to restore the EET of *S. oneidensis* MR-1, which couples arsenic concentration in the environment with the ability of *S. oneidensis* MR-1 for Fe(Ⅲ) or anode reduction. We conducted the Fe(Ⅲ) reduction experiment and found that the Fe(Ⅲ) reduction rates of Δ*mtrCptpA*/MtrC and Δ*cymAptpA* /CymA exhibited faster trend with the increase in As3+ concentration (Figure 5B and C). Moreover, when MFCs reached a maximum voltages, we added As3+ with the concentration of 100 μM and observed an increased in voltages of Δ*mtrCptpA*/MtrC and Δ*cymAptpA* /CymA MFCs. All results suggested that As3+ induced the expression of *mtrC* and *cymA* to recover EET of Δ*mtrCptpA*/MtrC and Δ*cymAptpA* /CymA, respectively.

![Pulpit rock](https://static.igem.wiki/teams/4767/wiki/project-results2/res-5.png)

Figure 5. (A) The schematic illustration of Part 3 and Part 4 in which the arsenic responsive transcription *arsR* - P*ars* system controls the expression of EET-associated gene *cymA* and *mtrC*, respectively. The Fe(Ⅲ) reduction of Δ*mtrCptpA*/MtrC (B) and Δ*cymAptpA* /CymA (C) incubated with different concentrations of As3+. The voltage of Δ*mtrCptpA*/MtrC (D) and Δ*cymAptpA* /CymA (E) MFCs fed 50 and 100 μM As3+ at 25 h.

## 3. Construct an Amplifier System for Detecting a Lower Concentration of Arsenic

To further improve the sensitivity of our biosensor, we introduced a positive feedback loop using the LuxR autoregulatory elements to arsenic EAB-based biosensor. As Figure 6A shows, the transcriptional activator, a variant of LuxR, was used to replace *mtrC* or *cymA* in Part 3 and Part 4, and it was regulated by the *arsR*-P*ars* circuit, while *mtrC* or *cymA* together with a second *luxR* was placed under the promoter P*luxI*, which was activated by LuxR. When arsenic is present, it activates the expression of the first LuxR, which turns on the expression of *mtrC* (or *cymA*) and the second LuxR. The second LuxR activates its own expression as well as that of *mtrC* (or *cymA*) and forms a positive feedback loop to enhance the output signal. The gene circuits were named as Part 8 and Part 9. Then, the Part 8 and Part 9 were transformed into Δ*mtrCptpA* and Δ*cymAptpA*, resulting in Δ*mtrCptpA*/MtrC(+) and Δ*cymAptpA*/CymA(+), respectively. We conducted the Fe(Ⅲ) reduction experiments and found that the Fe(Ⅲ) reduction rates of Δ*mtrCptpA*/MtrC(+) and Δ*cymAptpA*/CymA(+) were enhanced with an increase in As3+ concentrations (Figure B and C). It indicated that As3+ can induce the expression of *cymA* and *mtrC*. Comparation of the Fe(Ⅲ) reduction rates between Δ*mtrCptpA*/MtrC without the amplifier system (+) and Δ*mtrCptpA*/MtrC(+) with the amplifier system showed that Fe(Ⅲ) reduction rates of Δ*mtrCptpA*/MtrC(+) were significantly faster than those of Δ*mtrCptpA*/MtrC when 10 and 100 μM As3+ were added in the system (Figure 6D). Similar result was observed for Δ*cymAptpA*/CymA and Δ*cymAptpA*/CymA(+) strains (Figure 6E). All results suggest that the response of the positive feedback amplifier biosensor to As(Ⅲ) was faster, and the expression of MtrC and CymA were higher than those of the ones without positive feedback.

![Pulpit rock](https://static.igem.wiki/teams/4767/wiki/project-results2/res-6.png)

Figure 6. (A) The schematic illustration of Part 8 and Part 9 that are As(Ⅲ) biosensors with an amplifier circuit. The Fe(Ⅲ) reduction of Δ*mtrCptpA*/MtrC(+) (B) and Δ*cymAptpA*/CymA(+) (C) at different concentrations of As3+. The Fe(Ⅲ) reduction rate of Δ*mtrCptpA*/MtrC and Δ*mtrCptpA*/MtrC(+) for 26 h (D), as well as Δ*cymAptpA*/CymA and Δ*cymAptpA*/CymA(+) (E) with different concentrations of As3+ for 31 h. A two-sided Student’s t test was used in (D) and (E) to analyze the statistical significance (NS: No significance. \* 0.01< P < 0.05).

To test whether Δ*mtrCptpA*/MtrC(+) and Δ*cymAptpA*/CymA(+) biofilms as a bio-element can improve the sensitivity of EAB-based biosensors, As3+ with the concentrations of 50 and 100 μM was fed into the systems after MFCs generated stable voltages. We found that the presence of As3+ (50 and 100 μM) in the system increased the voltages of both Δ*mtrCptpA*/MtrC(+) and Δ*cymAptpA*/CymA(+) MFCs (Figure 7A and B). We compared the enhanced rate (ER) of voltages of Δ*mtrCptpA*/MtrC and Δ*mtrCptpA*/MtrC(+), and found that the enhanced rates of voltages of Δ*mtrCptpA*/MtrC(+) with an amplifier circuit were significantly higher than those of Δ*mtrCptpA*/MtrC when the concentrations of As3+ were 50 and 100 μM (Figure 7C). ER is used to evaluate the voltage promotion rate of the MFC. It is calculated as follows:
ER(%) = (Vm - V0)/V0 × 100%
where V0 is the maximum stable voltage before feeding arsenic, and Vm is the maximum voltage inducted. We observed the similar results for the Δ*cymAptpA*/CymA and Δ*cymAptpA*/CymA(+) strains. The results show that amplifier system improved the performance of EAB-based sensors.

![Pulpit rock](https://static.igem.wiki/teams/4767/wiki/project-results2/res-8.png)

Figure 7. The voltage of Δ*mtrCptpA*/MtrC (+) (A) and Δ*cymAptpA* /CymA(+) (B) MFCs fed 50 and 100 μM As3+ at 25 h. The enhanced rate of voltages of Δ*mtrCptpA*/MtrC and Δ*mtrCptpA*/MtrC (+) (C), as well as Δ*cymAptpA* /CymA and Δ*cymAptpA* /CymA(+) (D). A two-sided Student's t test was used in (C) and (D) to analyze the statistical significance . ( \*\* P < 0.01).

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