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: wild 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(final concentration=0.1mM) in it to induce KmAdh expression.
Figure 2. SDS-PAGE of KmAdh
(From left to right: wild type, KmAdh, KmAdh+IPTG)
From the SDS-PAGE result, it can be safely concluded that KmAdh was successfully expressed at a high level.
Then we gathered the bacteria to purify the KmAdh for enzyme activity measurement
Figure 3. SDS-PAGE of KmAdh
(From left to right: wild type, KmAdh, KmAdh+IPTG, raw enzyme, flow throgh, 20mM elution, 300mM elution, Pure enzyme)
From figure 3, you can see that we successfully purify the KmAdh from the bacteria lysate.
2.Enzyme activity test
NADH, as a necessary cofactor of KmAdh, has a significant absorption in 340nm. However, once it's been reduced to NAD+, it will have no absorption in 340 nm. So, 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
Here in figure 4 is how we performed the reaction. After adding every component in to the cuvette, we scan the 340nm UV absorption value over time. Because NADH is easy to be oxidized, we set a blank control to exclude this effect. The system is the same as the above system, with the same amount of PBS to replace KmAdh purified enzyme.
Figure 5. The change of OD340 over time
Here in figure 5, there is a 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.
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 wild type and KmAdh at diferent concentration of acetaldehyde and ethanol
Figure 7. The growth curve of wild type and KmAdh at diferent concentration of acetaldehyde and ethanol
4.Enzyme Activity Measurement in vivo
