Team:CHINA CD UESTC/Results
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RESULTS
Finally, 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 were something interesting appeared in the bacteria!
Vectors construction and activity detection of laccase
1. Amplification of target genes. We respectively amplified mamW, RFP and laccase by common PCR (Fig. 1A). In order to make laccase visible, we combined RFP with laccase. In order to immobilize laccase, we combined mamW+RFP+laccase and RFP+laccase by fusion PCR (Fig. 1B).
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. Verification of vectors. We purified the PCR products (Fig. 1B) and successfully inserted the fragments into pACYCDuet-1, and named piGEM-WRL and piGEM-RL respectively. We verified them using digestion (Fig. 2) 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. Transformation and inducible expression of RFP+laccase. We transformed the piGEM-RL into BL21(DE3) and conducted inducible expression. The color (Fig. 3A) and concentration (Fig. 3B) 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 OD600 of different concentration of baterium liquid at different time using ultraviolet spectrophotometer.
4. Test the expression of RFP+laccase. We broke the cells in boiling water for 10min and run SDS-PAGE with this sample (Fig. 4).
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.
In the gel we found that the band located near 84KDa is RFP+laccase.
5. Detect the activity of laccase. Crush the bacterium with Ultrasonic Cell Disruptor. Collect the supernatant (Fig. 5A) and detect the activity of laccase (Fig. 5B) 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 times.
6. Transformation and inducible expression of mamW+RFP+laccase. We transformed piGEM-WRL into BL21(DE3) and conducted inducible expression. We got a series of samples which was cultivated at different times and chose one to compare with BL21(DE3) untransformed (Fig. 6A). Then, we detected the activity of laccase (Fig. 6B).
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.
7. The activity comparation of two laccases. We compared the two enzyme activity curves(Fig. 7).
Figure 7. The activity comparation of two laccases. The red curve represented piGEM-RL and the blue curve presented piGEM-WRL.
From Fig. 5,6,7, we can see that both of the two fusion proteins have catalytic activity, while the activity of laccase, coded by piGEM-RL, is higher than another. It may caused by the concentration of the laccases. Next we put the laccases in the EBFC. We firstly put RFP+laccase into our EBFC.
Construction and test of EBFC
1. The sample EBFC 1.0. We made this device using discarded bottles with the proton exchange membrane in the middle (Fig. 8A). 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 (Fig. 8B).
Figure 8. The simple EBFC 1.0 made by ourselves.
Due to the fact that 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. The improved EBFC 2.0. We purchased the device from the internet. The middle of the device is proton exchange membrane, and the electrode material is carbon paper (Fig. 9A). We assembled the materials above and made the EBFC 2.0 (Fig. 9B).
Figure 9. The diagram of the EBFC 2.0.
EBFC performances. We added components into the device and test the performance with oscilloscope (Fig. 10).The figure 10A and B were control.
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 small electric current, so we need to further improve the battery.
3. The improved EFBC 3.0. In order to reduce the internal resistance and lower the cell cost, we designed the device 3.0 (Fig. 11).
Figure 11. The device made by 3D printer. Add the components through the upper holes and fix the electrodes to the edge of the holes.
Run the 3D printing 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 gathered on the surface of two electrodes and immobilized.
Next, we put MamW+RFP+laccase into our EBFC, the work is going on and detailed results may be presented on our PPT.
Magnetosome formation in E.coli
1. Amplification of mamAB. We separated mamAB into 3 parts and amplified each one by common PCR (Fig. 12A). We subcloned the three parts into pET-28a vector successfully, and named piGEM-AB. We verified it using digestion (Fig. 12B) 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. Amplification of mamGFDC+mms6 and mamXY. We also amplified mamGFDC+mms6, and mamXY by common PCR (Fig. 13A). We cloned the two parts into pCDFDuet-1 vector successfully, and named piGEM-G6X. We verified it using digestion (Fig. 13B) 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. Verification of co-transfermation.We constructed the piGEM-AB and piGEM-G6X successfully and co-transferred them into E.coli, and detected by the colony PCR (Fig. 14).
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) micrographs of cells. After we co-transferred the two vectors successfully, we attempted to attract the modified E.coli with magnet. But we didn’t observe the E.coli attracted by a permanent magnet at the edge of a culture flask. So we observed the E.coli under the TEM (Fig. 15).
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 200nm.
From Fig. 14, we can see that we co-transformed piGEM-AB and piGE-G6X into E.coli successfully. After further culture in large scale, we observed cells with TEM. From Fig. 15 we can see that E.coli’s shape changing with the increase of operons. After transforming all of the four operons, we saw black particles like megnetosomes. But we learned that inclusion bodies are also black particles when observing under a TEM. So we ran SDS-PAGE, however we didn’t find any specific band, which indicated the black particles (Fig. 15F, G, H) could well be magnetosomes. We further observed that E.coli with these black particles were mostly in the decline stage. So we need to do more experiments, on the one hand, to confirm whether the black particles are magnetosomes, and on the other hand, to make E.coli produce magnetosomes stably. Then we can use these magnetosomes to realize laccases’ enrichment and immobilization.