Team:CHINA CD UESTC/Results
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RESULTS
Fortunately, we got the fusion proteins MamW + RFP + Laccase and RFP + Laccase. The fusion protein RFP + Laccase worked very well in our EBFC. What's more, after we co-transferred the two vectors piGEM-AB and piGEM-G6X into escherichia coli BL21(DE3), there are something interesting appeared in the bacteria!
Laccase
1. We respectively amplified mamW, RFP and laccase by common PCR and then we respectively combined W+R+L and R+L by fusion PCR.
Figure 1. The image of agarose gel electrophoresis. (A) M: DNA marker, mamW and RFP and laccase were amplified by PCR using high fidelity DNA polymerase. (B) M: DNA marker, W+R+L: mamW+RFP+Laccase, R+L: RFP+ Laccase.
2. We purified the PCR products and successfully inserted the fragments into pACYCDuet-1, and named piGEM-WRL and piGEM-RL. We verified them using digestion and sequencing.
Figure 2. Verification of vectors using digestion. (A) M: DNA marker. Lane 1, the piGEM-WRL without restriction endonuclease. Lane 2, the piGEM-WRL was digested by PstI +XhoI. (B) M: DNA marker. Lane 3, the piGEM-RL without restriction endonuclease. Lane 4, the piGEM-RL was digested by PstI +XhoI.
3. We transformed the piGEM-RL into BL21(DE3) and conducted inducible expression. The concentration and color of bacterium liquid are as followed:
Figure 3. The color and the concentration of bacterium liquid. (A) The color degree among different bacterium liquid. The higher concentration of bacterium liquid showed redder. (B) The OD 600 of different concentration of baterium liquid at different time using ultraviolet spectrophotometer.
4. The SDS-PAGE of the cell with the piGEM-RL.
Figure 4. Testing expression of RFP+Laccase in E.coli. M: marker. Lane 1, bacteria untransformed. Lane 2, Bacteria which contain piGEM-RL. Induced at 37centigrades, 180rpm, 10 hours with 0.5mM IPTG.Figure 1.4: We confirmed piGEM-G6X by enzyme digestion using Hind III enzymes in Lane1 and Apal I and Pst I enzymes in Lane2.
In the gel we found that the band located near 80KDa is RFP+Laccase.
5. The activity of laccase：
Using Ultrasonic Cell Disruptor to crush the bacterium in ice-bath. Collect the supernatant and detect the activity of laccase by ABTS method.
Figure 5. The color of supernate and activity of laccase. (A) The higher concentration of laccase showed redder. (B) The activity of RFP+Laccase. Ultrasonic Cell Disruptor to crush the bacterium in ice-bath. Collect the Supernatant and detect the activity of laccase by ABTS method. The 1mL supernate equal to the 5mL bacterium liquid which were cultivated for different time.
6. We transformed piGEM-WRL into BL21(DE3) and conducted inducible expression. We got a series of samples which was cultivated at different time and chose one to compare with BL21(DE3) untransformed.
Figure 6. The color of supernate and activity of laccase. (A) The left is BL21(DE3) untransformed and the right is BL21(DE3) transformed with piGEM-WRL. (B) The activity of MamW+RFP+Laccase.
In the end, we compared the two enzyme activity curves.
Figure 7. The comparation of two laccases activity. The red curve represented piGEM-RL and the blue curve presented piGEM-WRL.
This picture showed that the enzyme activity of the laccase coded by piGEM-RL is higher than another. Next we put the RFP+Laccase in the EBFC.
Enzymatic biofuel cell (EBFC)
1. Device 1：EBFC 1.0
Figure 8. The simple EBFC 1.0 made by ourselves.(A) Make this device using discarded bottles with the proton exchange membrane in the middle.(B)Fix the EBFC 1.0 in the foam board, add each component (10 ml in total) into the device according to the protocol. Use multimeter and oscilloscope to test voltage.
Due to the fact that the sealing was not very good and there existed leakage phenomenon, after several tests, the voltage is basically 0V. After a resistance measure by the multimeter, we found that the battery’s internal resistance is too large, so we need to further improve the device.
2. Device 2: EBFC 2.0
Figure 9. The diagram of the EBFC 2.0. Purchase the device from the internet.(A)The middle of the device is proton exchange membrane; the electrode material is carbon paper.(B)The diagram of the fixed EBFC 2.0.
Figure 10. The EBFC 2.0 performances.(A)Before adding enzyme, the voltage was 0V.(B)the scan map of oscilloscope of the left.(C)After adding enzyme, the voltage increased instantly but the voltage was far from stable. After about 5 minutes, the voltage got stable.(D)The voltage reached a stable level of about 160mV and lasted for about 40h. The scan time was 50s and every grid represents 100mV.
From the Figure 10, we can see that the EBFC effected the desired result—voltage appearred. The voltage reached a stable level of about 160mV and lasted for about 40h. The highest voltage reached 0.25V. But the battery’s internal resistance was too large, resulting in the small electric current, so we need to further improve the battery.
3. Device 3 EFBC 3.0
Figure 11. The device made by 3D print. Add the components through the upper holes and fix the electrodes to the edge of the holes.
Run the 3D print device and test, it didn’t achieve the desired result; the internal resistance of the battery didn’t get smaller. Apart from reducing the internal resistance, we have to improve the electrons transfer efficiency and make enzyme catalyze substrates constantly. So we need to find a better method to make enzyme gather to the surface of two electrodes and immobilized.
Magnetosome
1. We separated mamAB into 3 parts and amplified each one by common PCR. We subcloned the three parts into pET28a vector successfully, and named piGEM-AB. We verified it using digestion and sequencing.
Figure 12. The construction of piGEM-AB. (A) The three parts of mamAB by PCR using high fidelity DNA polymerase. M: marker. Lane 1, mamAB part1. Lane 2, mamAB part2. Lane 3, mamAB part3.(B)M: marker. Lane 4, digestion the plasmid piGEM-AB by restriction endonuclease ApaI,SapI,NotI.
2. We also amplified mamGFDC+mms6, and mamXY by common PCR. We cloned the two parts into pCDFDuet-1 vector successfully, and named piGEM-G6X. We verified it using digestion and sequencing.
Figure 13. The construction of piGEM-G6X.(A) amplify mamGFDC+mms6 and mamXY by PCR using high fidelity DNA polymerase. M: marker. Lane 1, mamGFDC+mms6. Lane 2, mamXY. (B) M: marker. Lane 3, digestion the plasmid piGEM-G6X by restriction endonuclease HindIII. Lane 4, digestion the plasmid piGEM-G6X by restriction endonuclease ApalI, PstI.
3. We constructed the piGEM-AB and piGEM-G6X successfully and co-transferred them into E.coli, and detected by the colony PCR.
Figure 14. The bacterial colony PCR result. M: marker. Lane 1, the colony with piGEM-AB. Lane 2 and 3, the colony with piGEM-AB+piGEM-G6. Lane 4 and 5, the colony with piGEM-AB+piGEM-G6X.
4. Transmission electron microscope (TEM)
Figure 15. Transmission electron microscopy images of modified E.coli. (A) (B)Images of cells of BL21(DE3) without any vectors prepared on a TEM grid.(C) Images of cells of BL21(DE3) transferred with piGEM-AB.(D) Images of cells of BL21(DE3) co-transferred with piGEM-AB and piGEM-G6. (E)-(H) Images of cells of BL21(DE3) co-transferred with piGEM-AB and piGEM-G6X. Arrows indicate the magnetosome. The scale bar corresponds to 200 nm.