Team:ZJUT-China/Notebook
Human Practices
Notebook
Stage 1: gfa assembly and tests
We found glutathione-dependent formaldehyde-activiting enzyme which can catalyze degradation of formaldehyde through consulting literature materials. We use overlap to construct a pet28b plasmid with gfa and transform it in to BL21. Then we use formaldehyde solution to do a cotrolled experiment to see whether gfa works.
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Stage 2: transformation of lbfdh and test
We thought that the accumulation of too much HCOOH may do harm to the cells itself in affecting its pH and many other aspects. Thus, we add the gene lbfdh to the cell and test its effection.
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Stage 3: formaldehyde degradation assembly and tests
We consult literature materials to find out the reason why our engineered bacteria with gfa didn’t perform better. And we found that when gfa catalyze HCHO combine with GSH, it is better to add another two enzyme to turn HCHO into HCOOH. What’s more, with the improved serious of reactions, GSH acts as one of the catalysts and won’t be consumed.
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Stage 4: positive feedback system assembly and test
Considering the leakage of FrmR promoter and the link of formaldehyde degradation and color reaction indicator, we use the positive feedback system constructed with luxR and luxI. At the same time, we can connect the degradation, indication and concentration of formaldehyde together by the positive feedback system.
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To achieve this goal, we used the part one, which had been constructed by 2017 iGEM team ZJUT-China. And a series of improvements have been made to the system. Firstly, we use Lactose operon and negative induction system to establish a repression device. Next, we choose the lysis gene to achieve autolysis of bacteria.
After the successful construction of the system, we tested the efficiency of lysis gene expression by controlling the light intensity.
The experiment is divided into 3 steps:
1. Pre experiment: measure the expression efficiency of cleavage genes.
2. We use PCR technology to get the required fragments and connect these fragments.
3. We detect expression efficiency by measuring OD value.
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To achieve this goal, we constructed the color reaction based indicator system. The experiment is divided into following steps.
1. Selection of chromogenic agents.
2. Test the efficiency of pFrmR.
3. Construct the system by using pcr technology.
4. Quantify the relationship between the components in the pathway of Color Reaction Based Indicator System.
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