iGEM 2022 — Team xhd-wuhan-china


XHD-Wuhan-China

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Preliminary design

In order to develop an effective and environmentally friendly copper in-situ analysis method as an implementation tool for water quality assessment. Escherichia coli BL21 is genetically modified to express ribB with copper-sensitive promoter pcutR, which can sense copper ion concentration to produce riboflavin. Riboflavin can promote electron transfer to increase the voltage in microbial fuel cells.

Construction

Biobrick-compatible restriction site sequence was introduced into pET-23b (+) plasmid by inactivation kinase ligation kit, and named pLZ01 for standard component assembly. We synthesized copper-sensitive promoter and named pcutR by DNA synthesis, and synthesized ribB gene by DNA synthesis.

![](https://static.igem.wiki/teams/4172/wiki/engineer/pic1.png)

Escherichia coli BL21 carrying plasmid pLZ01-pcutR-GFP was constructed to test the sensitivity and specificity of pcutR.

![](https://static.igem.wiki/teams/4172/wiki/engineer/pic2.png)

Escherichia coli BL21 carrying plasmid pLZ01-pcutR-ribB was constructed to test the performance of the working system.

Test

Escherichia coli BL21 carrying plasmid pLZ01-pcutR-GFP was cultured overnight at 160rpm in a shaking table at 37 ℃. The cultured Escherichia coli was inoculated in solutions containing different concentrations of copper ions (0, 0.05, 0.2, 0.5, 1, 1.2 mM) for 4 hours. Then, the fluorescence intensity of GFP (excitation wavelength: 490 nm; emission wavelength: 510 nm) and OD600 of Escherichia coli BL21 carrying plasmid pLZ01-pcutR-GFP were measured by microplate reader. The average GFP/cell was calculated by dividing the original fluorescence intensity by OD600. In order to observe the expression of GFP in cells induced by copper ion, the culture was centrifuged (5000 g, 5 min, 4 ℃) and suspended again in PBS buffer (10 mM, pH 7.4) after 4 hours of culture. Fluorescence imaging was performed on a fluorescence microscope (Olympus, Japan) using a 40-fold objective lens.

![](https://static.igem.wiki/teams/4172/wiki/engineer/pic3.png)

Fluorescence microscopy image of above reporter BL21 and control exposed to 0.5 mM Cu2+

![](https://static.igem.wiki/teams/4172/wiki/engineer/pic4.png)

E. coli BL21 containing reporter plasmid pLZ01-pcutR-GFP was incubated with various CuSO4 for 4h to determine the concentration dependence of pcutR to Cu2+

The results showed that copper ion could initiate the expression of green fluorescent protein GFP in copper sensitive promoter pcutR, and the initiation degree of copper sensitive promoter pcutR was linearly related to copper ion concentration

The recombinant plasmid pLZ01-pcutR-ribB was transferred to Escherichia coli BL21 for expression.

A two-chamber MFC reactor with a working volume of 240 mL was set up, and the electrodes were pretreated before use. Carbon felt with an area of 16cm2 was used as anode and cathode. These electrodes are connected by titanium wires to an external resistor of 1000 Ω.

In MFC operating system, different concentrations of Cu2+ (0-500μM) were added to the anode medium in M9 liquid medium for Cu2+ response regulator use. The cathode solution was potassium ferricyanide (100 mM potassium ferricyanide in 50 mM phosphate buffer, pH 7.0). Voltage was recorded at 10 min intervals in an MFC biosensor using a data acquisition device.

![](https://static.igem.wiki/teams/4172/wiki/engineer/pic5.png)

Relationship between copper ion concentration and the maximum voltage of the constructed MFC biosensor

![](https://static.igem.wiki/teams/4172/wiki/engineer/pic6.png)

Comparison of the maximum voltage between engineered and wild bacteria at 500 μm Cu ion concentration

The results showed that the expression of ribB gene promoted electron transfer and eventually led to a significant increase in MFC voltage, and there was a linear relationship between ribB concentration and MFC voltage

Learning

Escherichia coli has a compact and low permeability membrane, which limits the transfer of electrons from cells to electrodes. When we went to Huada for gene exchange, relevant experts suggested that porin-related genes could enhance the permeability of Escherichia coli cell membrane, so we consulted a large number of literatures and found that oprF porin gene could increase the permeability of Escherichia coli cell membrane and enhance extracellular electron transfer and bioelectric output.

Redesign

The porin gene (oprF) was synthesized by direct DNA synthesis and regulated by constitutive promoter to ensure the continuous expression of the gene.

Construction

![](https://static.igem.wiki/teams/4172/wiki/engineer/pic7.png)

We reconstructed our genetic circuit map. T7 Promoter (constitutive promoter), oprF porin gene, pcutR copper sensitive promoter and ribB riboflavin synthesis gene were combined together.

![](https://static.igem.wiki/teams/4172/wiki/engineer/pic8.png)

T7 Promoter (constitutive promoter), oprF porin gene constitute our amplification system; The pcutR copper-sensitive promoter and ribB riboflavin synthesis gene constitute our working system.

Finally, we constructed our recombinant plasmid pLZ01-pcutR-ribB-oprF

Test

The recombinant plasmid pLZ01-pcutR-ribB-oprF was transferred to Escherichia coli BL21 for expression.

A two-chamber MFC reactor with a working volume of 240 mL was set up, and the electrodes were pretreated before use. Carbon felt with an area of 16cm2 was used as anode and cathode. These electrodes are connected by titanium wires to an external resistor of 1000 Ω.

In MFC operating system, Zn2+ with different concentrations (0-500 μ M) is added to the anode medium in M9 liquid culture medium, so that Zn2+ can be used in response to the regulator. The cathode solution was potassium ferricyanide (100 mM potassium ferricyanide in 50 mM phosphate buffer, pH 7.0), and voltages were recorded at 10 min intervals in an MFC biosensor using a data acquisition device.

![](https://static.igem.wiki/teams/4172/wiki/engineer/pic9.png)

Relationship between copper ion concentration and the maximum voltage of the constructed MFC biosensor

![](https://static.igem.wiki/teams/4172/wiki/engineer/pic10.png)

Comparison of the maximum voltage between PLZ01-PcutR-RIBB-OPRF engineered bacteria and PLZ01-PCUTR-RIbb engineered bacteria at 500 μM copper ion concentration

The experimental results showed that oprF porin gene significantly changed the permeability of cell membrane, and its amplification effect was about 6 times. In the modified MFC sensor, there is a linear relationship between copper ion concentration and maximum voltage.

Conclusion

We have made a microbial sensor for copper contamination detection with simple detection process and suitable for real-time monitoring. The introduction of oprF porin gene greatly improves the detection performance of our sensor.

Reference

Yong, Y.-C., Yu, Y.-Y., Yang, Y., Liu, J., Wang, J.-Y. and Song, H. (2013), Enhancement of extracellular electron transfer and bioelectricity output by synthetic porin. Biotechnol. Bioeng., 110: 408-416.

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