iGEM 2022 — Team xhd-wuhan-pro-china


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{% block title %}Engineering Success{% endblock %}
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Equipment design

Make better and more efficient equipment to monitor the degree of pollutant emission, and design a microbial sensor, the sensor of microbial sensor is a kind of membrane containing microorganisms. The sensor works with microorganisms that consume the dissolved oxygen in the solution to be tested and then release heat or light to achieve the purpose of quantifying the detected substance. It will also use microbial fuel cells, a device that uses microbes to convert chemical energy from an organic matter directly into electricity. Because we consider that microbial fuel cells can be used in many fields such as power generation, biological hydrogen production, biosensors, and wastewater treatment. Because of its low cost, uniqueness and efficient, strong adaptability, and other advantages, so the current development prospects are excellent. The basic working principle is the equipment in the anode chamber under the anaerobic environment, the microbes break down organic matter and release of electrons and protons, electrons depend on appropriate electron transfer between the anode and the biological components for effective electron transfer, through the proton exchange membrane to the cathode, oxidant (generally for oxygen) in the heart of the cathodic reduction of electrons and protons combine to form water.

In the early stage of device design, we combined the pzntR zinc-sensitive promoter, RBS ribosome binding site, ribB riboflavin synthesis gene, and Terminator Terminator, and constructed plasmids pUC19 and Petduet-1 using molecular biology tools. Zinc-responsive promoter (pzntR) and riboflavin synthesis gene (ribB) were synthesized by direct DNA synthesis method. PzntR and GFP were ligated. The fragment was digested with Hind ⅲ and BamHⅰ and inserted into plasmid pUC19 to recombine, which was named PUC19-PZNTR-GFP. Then it was transferred to E.coli BL21 for expression.

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Subsequently, engineered E.coli BL21 strains and wild-type E.coli BL21 strains were cultured in LB liquid medium at 37℃, and different concentrations (0, 30, 60, and 90 μM) of Zn2+ were added at OD600 of 0.6, respectively. Three hours after the addition of Zn2+, engineered cells and wild cells induced by Zn2+ were observed under a fluorescence microscope. A 96 Well Plate Reader was used to measure GFP (the excitation wavelength of GFP was 488nm, and the emission wavelength was 507nm).

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The recombinant plasmid was transferred to Escherichia coli BL21 for further expression. A two-compartment MFC-operated reactor was set up with a working volume of 240 mL in each chamber, and the electrodes were pretreated before use. Carbon felt with an area of 16cm2 is used as an anode and cathode. These electrodes are connected via titanium wires to a 1000-ω external resistor. In the MFC operating system, the anodic medium was supplemented with different concentrations (0-500 μm) of Zn2+ in an M9 liquid medium for use by the Zn2+ response regulator. The cathode solution was potassium ferricyanide (100 mM ferricyanide in 50 mM phosphate buffer, pH 7.0), and the voltage was recorded at 10-min intervals in an MFC biosensor using a data acquisition device.

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Finally, a significant linear relationship was found between Zn2+ concentration and the maximum voltage of the constructed MFC biosensor.

By t-test of the maximum voltage of engineered bacteria and wild bacteria at the concentration of 500 μmZn 2+, P < 0.001, it indicates that riboflavin synthesized by ribB riboflavin synthesis gene can significantly promote electron transfer, which finally proves that our system can work normally.

![](https://static.igem.wiki/teams/4155/wiki/engin/img5.png)

Then, to improve the capability of the device, T7 Promoter is the constitutive Promoter (which maintains continuous activity in most or all tissues), the RBS ribosome binding site, the oprF porin gene Terminator, the pzntR Zn-sensitive Promoter, the ribB riboflavin synthesis gene.

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We built it into two systems:

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Direct DNA synthesis of the porin gene (oprF) The recombinant plasmid was transferred into Escherichia coli BL21 for further development Line. A two-compartment MFC-operated reactor was set up, and the working volume of each compartment was 240 mL. The electrodes were pretreated before use. Carbon felt with an area of 16cm2 is used as an anode and cathode. These electrodes are connected via titanium wires to a 1000-ω external resistor. In the MFC operating system, the anodic medium was supplemented with an M9 liquid medium at different concentrations (0-500 μm) of Zn2+ for use with the Zn2þ response regulator. The cathode solution was potassium ferricyanide (100 mM ferricyanide in 50 mM phosphate buffer, pH 7.0), and the voltage was recorded at 10-min intervals in an MFC biosensor using a data acquisition device.

![](https://static.igem.wiki/teams/4155/wiki/engin/img8.png)

After improvement, we found that there was a significant linear relationship between Zn2+ concentration and the maximum voltage of the constructed MFC biosensor. Moreover, the expression of oprF in the engineered strain BL21 increased the permeability of cell membrane, and the highest voltage was about 4.5 times higher than that of another engineered strain (Petduet-1PZnTR-Ribb).

![](https://static.igem.wiki/teams/4155/wiki/engin/img9.png)

After that, we compared the two engineered bacteria at the concentration of 500μMZn2+ by t test of the maximum voltage of engineered bacteria at the concentration of 500μMZn2+, P < 0.001, indicating that oprF gene significantly changed the permeability of cell membrane.

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