proof-of-concept
Pathway and working principle
In the presence of copper ion, copper-sensitive promoter pcutR is activated, ribB gene is expressed, which promotes riboflavin synthesis, and riboflavin promotes electron transfer to generate voltage. With the help of porin encoded by oprF, it promotes electron transfer and increases the voltage generation of MFC.
Evaluation on copper ion dependence of pcutR
Schematic illustration
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 a copper-sensitive promoter by DNA synthesis and named it pcutR.
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.
Fluorescence microscopy image of above reporter BL21 and control exposed to 0.5 mM Cu2+
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
Working system verification
Schematic illustration
We synthesized ribB gene by DNA synthesis. 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
Relationship between copper ion concentration and the maximum voltage of the constructed MFC biosensor
Comparison of the maximum voltage between engineered and wild bacteria at 500μm Cu ion concentration
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
Amplification system verification
Schematic illustration
Synthesis of porin gene (oprF) by direct DNA synthesis
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.
Relationship between copper ion concentration and the maximum voltage of the constructed MFC biosensor
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.
Summary
The recommended safety standard of copper ion concentration in drinking water is 31μM, which can be detected by us. Therefore, the biosensor can be used as an early warning device to ensure the drinking water safety of residents in cities or remote areas.
To sum up, we provide an economical, sensitive and practical technology for the detection of copper ions in water.