Team:USTC/Results
Electron Transfer System
1. DNA sequencing result and colony PCR
In order to verify the plasmid that we constructed, we designed 8 sequence-primers AF, AR, BF, BR, CF, CR, VF, VR and sequenced our plasmid, each one for 1000bp. The results are laid in appendix/DNA_sequencing/AF~VR.
Here is the result of plasmid extraction and colony PCR. Both are positive.
Figure 1. The result of plasmid extraction and colony PCR
2. Microbial Fuel Cell(MFC)
MFC device consists of cathode cell and anode cell separated by cation exchange membrane and carbon felt soaked in cathode solution and anode solution respectively. The voltage between the two carbon felt can be read by the detection device connected to the carbon felt.
When the bacteria that can express Mtr protein attached to the carbon felt at the cathode, Mtr transmit electrons to the anode through the carbon felt. The protons in the solution also pass through the cation exchange membrane to form a circuit and generate voltage between the cathode and anode of the battery. So the voltage is positively related to the ability of bacteria to release electrons.
We added K3[Fe(CN)6] cathode solution to the anode of MFC, added aerobic mineral salt culture medium to the cathode, aerated the cathode with nitrogen and connect the monitoring device. Then, we recorded the voltage between the positive and negative electrodes every ten minutes after the bacterial solution was injected. The raw data are laid in appendix/MFC. After ploted with the voltage as the ordinate and the time as the abscissa, we got this graph.
Figure 2. MFC
The diagram of the experimental results shows that the voltage produced by MTR overexpression strain is stronger than that produced by empty plasmid strain, which shows that MTR overexpression brings stronger electron releasing ability to S. oneidensis MR-1.
3. Reduction of MO
The raw data are laid in appendix/reduction_of_MO/… . After necessary integration, we got an excel as follows.
Figure 3. Reduction of MO
Well H1~H8 were injected for MO concentration standard curve. After linear fitting, we got the relation between OD465 and the concentration of MO.
Figure 4.
According to this equation, we got the change of MO concentration of each group as time went by. Then, we calculated the average concentration as final data and standard deviation as error bar. After exponential fitting, we got this graph.
Figure 5.
The exact reduction rate makes little sense because the incubation condition and original concentration of azo dye are far from identical between lab experiment and factory practice. The relative improvement, however, can prove that our engineered strain is a successful system to reduce azo dye. From the fitting equations, we can get that the ratio of slopes at the same MO concentration is k=bExp/bWT=0.01362/0.0014=9.73. Thus, considering wildtype S. oneidensis MR-1 itself is a wonderful “azo-dye-discolor strain”, we did construct a very powerful azo dye reduction system with its amazing reduction efficiency as about 10 times higher as the original strain.
4. Verification of QS system
In order to verify our QS system, we have done the QS verification experiment (you can click here to see how we did the experiment).
We measured bacterial concentration (OD600 of 200μL medium in GREINER 96 F-BOTTOM) and the GFP intensity (excitation wavelength=485nm, emission wavelength=525nm, gain=800) of 2X YT bacteria medium (with 50ng/ml Kan), engineered S. oneidensis MR-1（with PYYDT-GFP plasmid）and wild type S. oneidensis MR-1(with empty PYYDT plasmid) with time. The raw data are laid in appendix/QS_verification/raw_data/… .
On the data sheet, we can see an interesting thing that the GFP intensity of WT is reducing with bacterial concentration growing. But it doesn’t matter, we use relative GFP intensity, whose value equal to the value of the GFP intensity of engineered S. oneidensis MR-1(with PYYDT-GFP plasmid)minus the value of the GFP intensity of wild type S. oneidensis MR-1(with empty PYYDT plasmid). We also use relative bacterial concentration, whose value is equal to the value of the OD600 of engineered S. oneidensis MR-1(with PYYDT-GFP plasmid)minus the value of the OD600 of YT bacteria medium (with 50ng/ml Kan).
We use MatLab software to manage the above process and deal with the data and make fitted curves. Firstly, we make fitted curves of relative concentration change with time(picture a) and relative GFP intensity change with time(picture b). After that, we calculated the slope of the curve of picture b as the relative value of expression and made it divided by the value of relative concentration at each time, to get the value of relative expression efficiency of GFP. Then, we make fitted curves of relative expression efficiency change of GFP with relative concentration (picture c). Here are the picture a, b, c and the codes we have programmed are laid in appendix/QS_verification/matlab_code/ PYYDT_GFP_code.
Figure 6. Verification of QS system
On the picture(c), we can see that when the concentration is low (OD600 lower than about 0.2), the relative expression efficiency of GFP is 0. And when the concentration is higher than a certain value (OD600>0.2), the relative expression efficiency of GFP starts to rise until the concentration reach another certain value (OD600=0.5). Therefore, we can draw a conclusion that our QS system works well.
aNAT
Construction of aNAT expression vector
We clone the gene and vector with primers aNAT-F-SWJN, aNAT-R-SWJN, aNAT-backbone-F-SWJN and aNAT-backbone-R-SWJN.
Figure 7. PCR result of vectors
Figure 8. PCR result of aNAT
The bacteria PCR result is as follows
Figure 9. The bacteria PCR result
Characterization of aromatic amine resistance
Table 1. The average OD600 of S.oneidensis
Figure 10. The growing curve of different types of S.oneidensis
The result significantly demonstrates that S.oneidensis with aNAT grows better in 40ug/mL sulfanilamide than wild type and empty pYYDT controlling groups. The shaking of aNAT and empty pYYDT growing curves is possibly because of the lack of nutrition in aerobic mineral salt medium, and the pressure of kanamycin makes the growth of bacteria with pYYDT unstable.