Difference between revisions of "Team:CHINA CD UESTC/Results"

We separated mamAB into 3 parts and amplified each one by PCR.

Figure 1.1: We amplified three parts of mamAB by PCR using high fidelity polymerase Advantage.

We used three parts to constructed mamAB successfully by subclone and we have verified them using digestion (Fig.1) and sequencing.

Figure 1.2: We confirmed mamAB by enzyme digestion using ApaI、SapI、NotI enzymes

We also constructed mamGFDC+mms6, and mamXY which are related to the formation of magnetosome.

Figure 1.3: We amplified mamGFDC+mms6 and mamXY by PCR using high fidelity polymerase Advantage.

This is part has relation with the formation of magnetosome

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.

We respectively amplified three fragments by PCR.

Figure 1.5: We amplified mamW and RFP and Laccase by PCR using high fidelity polymerase Advantage.

We successfully connected three fragments together by fusion PCR

Figure 1.6: We amplified mamW+RFP+Laccase and RFP+ Laccase by fusion PCR using high fidelity polymerase Advantage.

We recovered the PCR products and succeddfully inserted the fragment into pACYCDuet. and confirmed piGEM-WRL and piGEM-RL by restriction digestion.

Lane1 :control of pACYCDuet-1-RFP-Laccasse
Lane2 :BL21 Bacterial strain which contain pACYCDuet-1-RFP-Laccasse induced expression.

Figure 2.6: We choose one to compared with BL21 bacterial strain containing empty vector as control group and showed that experimental group is redder than control group.

Figure 2.7: We determined enzymes activities from different supernate using ABTS method. Every supernate presents the different bacterium which are cultivated for different time.

Figure 2.8: two enzyme activity curves presented the two different laccases coded by different vector. the red curve represented piGEM-RL and the blue curve presented piGEM-WRL.

Figure 4.3. The middle of the device is proton exchange membrane; the electrode material is carbon paper. Run the device.

Figure 4.4. Before adding enzyme, the voltage is 0; the right is the scan map of oscilloscope in the left.

Figure 4.8. 3D - print EBFC device.

Run the 3D print device and test, it didn’t achieve the desired result; the internal resistance of the battery didn't get smaller. In order to further improve the energy-producing effect and make enzyme catalyze substrates constantly, we need to find a better method to make enzyme gathered on the surface of two electrodes and fixed, so enzyme wouldn’t decrease with the change of substrates, realizing enzyme’s constant catalysis and improving the longevity of the battery.

Results of the promoter validation work:

In order to find the reason why magnetosomes canot form, we designed experiments to validate the promoters of 4 operons (connected to the design page).We successfully constructed the promoter validation work related vectors and transferred them to E. coli, cultured at 37 ℃and 180 RPM for 24 hours, but the bacteria liquid didn’t turn red. Then we broke cells and operate SDS-page electrophoresis, but we also didn’t find an RFP corresponding electrophoretic band. It meant that the native promoters related to the formation of magnetosomes in magnetotactic bacteria can’t work in E.coli, so finally we didn’t get magnetosomes.