Team:USTC/Demonstrate/3
1.Transformation and Expression
We transformed a plasmid PET22b containing KmAdh into E.coli successfully. We use KmAdh’s specific primers to do PCR to verify this achievement.
Figure 1. Electrophoresis result of PCR of KmAdh
(From left to right: wide type, KmAdh, positive control)
We can see that the experimental group and the positive control have the same band but WT does not. This shows the transformation is successful.
Then we induced the expression of this enzyme. We use 200 mL LB to cultivate our bacteria in 37℃，250 rpm. When its OD600 reached 0.5-0.8 we added 20μL 1M IPTG in it to induce KmAdh expression.
Figure 2. SDS-PAGE of KmAdh
(From left to right: wide type, KmAdh, KmAdh+IPTG)
We can see from the SDS page that KmAdh was successfully expressed.
Then we centrifuge the cells at 8000 rpm, 4°C, for 10 min and then remove the supernatant . Resuspend the bacteria with 50 mL PBS; Centrifuge at 8000 rpm, 4°C, for 10 min and then remove the supernatant. Resuspend the bacteria with 15 mL PBS. The cells were disrupted via ultrasonication (Power 30%, 30 min. Total duty in cycles of 1s on, 2s off). Finally we centrifuge it at 14000 rpm, 4°C, for 20 min and retrieve the supernatant for future purification. The crude enzyme is purified by nickel column to get the pure enzyme.
Figure 3. SDS-PAGE of KmAdh
(From left to right: wide type, KmAdh, KmAdh+IPTG, raw enzyme, flow throgh, 20mM elution, 300mM elution, Pure enzyme)
2.Enzyme activity test
NADH, as a necessary cofactor of KmAdh, has a significant absorption in 340nm. Along the process of the reduction reaction, the consuming of NADH will lead to a decrease of absorption in 340nm which allows us to test the activity of KmAdh by the spectrophotometer.
Figure 4. Reaction mixture
After adding, we scan the 340nm UV absorption value over time. Because NADH is easy to be oxidized, we set a blank control. The system is the same as the above system, but with the same amount of PBS instead of KmAdh.
We can obviously see the rapid increase in the absorption value after adding enzyme, indicating that NADH is drastically consumed. This shows the purified enzyme function is normal and the KmAdh is successfully expressed in E. coli.
Figure 5. The change of OD340 over time
3.Toxicity test
Considering that acetaldehyde and ethanol, the substrate and product of KmAdh, may do harm to the cell, we first made the growth curve of E.coli at different concentrations of acetaldehyde and ethanol to figure out a proper experimental condition. For acetaldehyde and ethanol, we both set four concentrations: 0%, 0.1%, 0.2% and 0.3%, and the results are shown in the following figures.
As the concentration of ethanol in the system increases, the growth of KMADH and WT is inhibited but KMADH’s growth is clearly better than WT’s at the same concentration. The reason is that KmAdh also has the effect of helping to break down ethanol. For acetaldehyde, the growth of KMADH and WT are both inhibited when the acetaldehyde concentration increases and KMADH’s growth is significantly better than WT’s when the acetaldehyde concentration reaches 0.3% (the highest concentration we set). This result is a rough proof of our KmAdh’s function is normal and the enzyme can be relatively high toxic acetaldehyde into less toxic ethanol to improve cell viability. When the acetaldehyde concentration and ethanol concentration in the system are the same, not only WT’s growth but also KMADH’s growth is inhibited. This indicates that the toxic effects of acetaldehyde on cells are stronger than ethanol.
According to the results, we decided to use 0.1% acetaldehyde as the substrate, for E.coli can live well.
Figure 6. The growth curve of wide type and KmAdh at diferent concentration of acetaldehyde and ethanol
Figure 7. The growth curve of wide type and KmAdh at diferent concentration of acetaldehyde and ethanol
4.Enzyme Activity Measurement in vivo
