Difference between revisions of "Part:BBa K2242626"
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| − | <h3>1.Transformation and Expression</h3> | + | <h3>1. Transformation and Expression</h3> |
<p>We transformed a plasmid PET22b containing KmAdh into <i>E.coli</i> successfully. We use KmAdh’s specific primers to do PCR to verify this achievement. </p> | <p>We transformed a plasmid PET22b containing KmAdh into <i>E.coli</i> successfully. We use KmAdh’s specific primers to do PCR to verify this achievement. </p> | ||
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<p>From figure 3, you can see that we successfully purify the KmAdh from the bacteria lysate. | <p>From figure 3, you can see that we successfully purify the KmAdh from the bacteria lysate. | ||
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| − | < | + | <h3>2. Enzyme activity test</h3> |
<p class="indent_word">NADH, as a necessary cofactor of KmAdh, has a significant absorption in 340nm. However, once it's been reduced to NAD<sup>+</sup>, 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. </p> | <p class="indent_word">NADH, as a necessary cofactor of KmAdh, has a significant absorption in 340nm. However, once it's been reduced to NAD<sup>+</sup>, 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. </p> | ||
<div style="text-align:center"> <img src="https://static.igem.org/mediawiki/2017/3/3a/USTC-result-harvest-6.jpeg" width="60%"> | <div style="text-align:center"> <img src="https://static.igem.org/mediawiki/2017/3/3a/USTC-result-harvest-6.jpeg" width="60%"> | ||
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<p class="indent_word">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. </p> | <p class="indent_word">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. </p> | ||
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| − | < | + | <h3>3. Toxicity test</h3> |
<p class="indent_word">Considering that acetaldehyde and ethanol, the substrate and product of KmAdh, may do harm to the cell, we first made the growth curve of <span class="italic">E.coli</span> 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. </p> | <p class="indent_word">Considering that acetaldehyde and ethanol, the substrate and product of KmAdh, may do harm to the cell, we first made the growth curve of <span class="italic">E.coli</span> 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. </p> | ||
<div style="text-align:center"> <img src="https://static.igem.org/mediawiki/2017/0/07/USTC-result-harvest-5%281%29.jpeg" width="60%"> | <div style="text-align:center"> <img src="https://static.igem.org/mediawiki/2017/0/07/USTC-result-harvest-5%281%29.jpeg" width="60%"> | ||
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<p class="indent_word">According to the results, we decided to use 0.1% acetaldehyde as the substrate, for <span class="italic">E.coli</span> can live well. </p> | <p class="indent_word">According to the results, we decided to use 0.1% acetaldehyde as the substrate, for <span class="italic">E.coli</span> can live well. </p> | ||
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| − | < | + | <h3>4.Enzyme Activity Measurement in vivo</h3> |
<p class="indent_word">As you can see above, this reductase KmAdh has strong enzyme activity in vitro. However, in the practical situation, we need this enzyme to function in vivo. So we did a enzyme activity measurement assay in vivo.</p> | <p class="indent_word">As you can see above, this reductase KmAdh has strong enzyme activity in vitro. However, in the practical situation, we need this enzyme to function in vivo. So we did a enzyme activity measurement assay in vivo.</p> | ||
<p class="indent_word">Here is how we performed this experiment. First, because the condition would be anaerobic when we are running the bio-cathode, so we simulate this anaerobic condition when we are measuring the enzyme activity. Figure 8 here shows how the system was constructed. Same procedure would be taken to create this anaerobic condition as how we did in the conduction system section.Then we put the anaerobic bottles to a incubator at 30˚C and added 0.1% ethanal(final concentration is 251 µmol/L) to initiate the reaction. </p> | <p class="indent_word">Here is how we performed this experiment. First, because the condition would be anaerobic when we are running the bio-cathode, so we simulate this anaerobic condition when we are measuring the enzyme activity. Figure 8 here shows how the system was constructed. Same procedure would be taken to create this anaerobic condition as how we did in the conduction system section.Then we put the anaerobic bottles to a incubator at 30˚C and added 0.1% ethanal(final concentration is 251 µmol/L) to initiate the reaction. </p> | ||
Latest revision as of 15:03, 29 October 2017
Alcohol dehydrogenases from Kluyveromyces marxianus
KmAdh is an alcohol dehydrogenase from Kluyveromyces marxianus. It can catalyze the ethanol fermentation ongoing in the bacteria with a high efficiency and mild reaction condition, especially temperature.
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 for 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 for 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.
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
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.
4.Enzyme Activity Measurement in vivo
As you can see above, this reductase KmAdh has strong enzyme activity in vitro. However, in the practical situation, we need this enzyme to function in vivo. So we did a enzyme activity measurement assay in vivo.
Here is how we performed this experiment. First, because the condition would be anaerobic when we are running the bio-cathode, so we simulate this anaerobic condition when we are measuring the enzyme activity. Figure 8 here shows how the system was constructed. Same procedure would be taken to create this anaerobic condition as how we did in the conduction system section.Then we put the anaerobic bottles to a incubator at 30˚C and added 0.1% ethanal(final concentration is 251 µmol/L) to initiate the reaction.
We took samples from the bottles 2 hour and 26 hour later and used Gas chromatography–mass spectrometry(GC-MS) to analyze the chemical compound in the sample, specifically, the concentration of ethanol and ethanal.
Before we use GC-MS to analyze the sample, we need to use standard sample to find out the appearance time for the compound we concerned. Here in figure are the results of standard samples for ethanol, ethanal and acetic acid( used as the internal standard to measure the concentration of ethanol and ethanal).
Figure 8. Result of standard sample of ethanol
Figure 9. Result of standard sample of ethanal
Figure 10. Result of standard sample of acetic acid
As you can see above, the appearance time for ethanol, ethanal, acetic acid are 3.047, 1.402, 7.109 respectively. The highest peak is the one of acetone, the solvent in our sample.
Then we began the analysis of the sample we had. Here is the outcomes of these 4 samples. After calculation, we had an accurate result of the concentration of ethanal in the system. However, the concentration of ethanol in the system is too low to be detected. So we could not measure the concentration of ethanol.
Figure 11. Result of GC-MS in table
Figure 12. Result of GC-MS in histogram
It’s true that the enzyme can catalyze the bio-transformation from ethanal to ethanol. In another word, the decrease of ethanal in the system should lead to the increase of ethanol. But it may be a chance that the ethanol had been used for metabolism. Because in the former procedures before we ran the bio-cathode, there was a step for starvation.The bacteria would be at a state that run out of carbon source, so the ethanol would become the carbon source and be consumed after it was synthesized. So we can not detect ethanol in the system.
Although we can’t detect the ethanol’s concentration to confirm whether the reductase has function in vivo, we can still compare the ethanal’s concentration to do the same work. As you can see in figure, the strain that expressed KmAdh utilized more ethanal than WT, which means this enzyme can increase the consumption speed of ethanal. This is a strong evidence to prove that this enzyme can function in vivo!
In conclusion, with GC-MS, we can confirm that the KmAdh can function in vivo under anaerobic condition.
Reference:
[1]Liang, J. J., Zhang, M. L., Ding, M., Mai, Z. M., Wu, S. X., Du, Y., & Feng, J. X. (2014). Alcohol dehydrogenases from Kluyveromyces marxianus: heterologous expression in Escherichia coli and biochemical characterization. BMC biotechnology, 14(1), 45.[2]Deng, M. D., Severson, D. K., Grund, A. D., Wassink, S. L., Burlingame, R. P., Berry, A., ... & Rosson, R. A. (2005). Metabolic engineering of Escherichia coli for industrial production of glucosamine and N-acetylglucosamine. Metabolic engineering, 7(3), 201-214.
[3]He, Y.C., Tao, Z.C., Zhang, X., Yang, Z.X., Xu, J.H., 2014a. Highly efficient synthesis of ethyl (S)-4-chloro-3-hydroxybutanoate and its derivatives by a robust NADH- dependent reductase from E. coli CCZU-K14. Bioresour. Technol. 161, 461–464.
[4]Cordell, R. L., Pandya, H., Hubbard, M., Turner, M. A., & Monks, P. S. (2013). GC-MS analysis of ethanol and other volatile compounds in micro-volume blood samples—quantifying neonatal exposure. Analytical and bioanalytical chemistry, 405(12), 4139-4147.
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