Part:BBa_K4172002

Added by XHD-Wuhan-China

Evaluation on copper ion dependence of pcutR

Figure 1 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.

Figure 2 Fluorescence microscopy image of above reporter BL21 and control exposed to 0.5 mM Cu2+

Figure 3 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

Figure 4 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

Figure 5 Relationship between copper ion concentration and the maximum voltage of the constructed MFC biosensor

Figure 6 Comparison of the maximum voltage between engineered and wild bacteria at 500μm 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

Amplification system verification

Figure 7 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.

Figure 8 Relationship between copper ion concentration and the maximum voltage of the constructed MFC biosensor

Figure 9 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.

CusS/CusR two component system+ribB+ oprF

The part uses the pCusC promoter which is sensitive to copper via the E. coli CusS/CusR two component system. The promoter responds directly to the CusR cytoplasmic response regulator which is activated by a transmembrane histidine kinase enzyme CusS found in the cell membrane. A standard ribosome binding site was added to the composite device after the promoter.The composite ends with a double terminator.A major limiting factor is the low bacterial membrane permeability, hindering transport of electron shuttles through the membrane and thereby restricting the electron shuttle-mediated extracellular electron transfer (EET) from bacteria to electrodes. This results in a reduced electrical power output of the MFC. Therefore, we heterologously expressed the porin protein OprF from Pseudomonas fluorescens into Escherichia coli.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1446
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 582
  Illegal AgeI site found at 1336
  Illegal AgeI site found at 1378
  Illegal AgeI site found at 2301
  
• 1000
  COMPATIBLE WITH RFC[1000]