Team:ZJUT-China/Design
Human Practices
Design
The goal of our project is to degrade formaldehyde. So we add 5 different protein aiming to degrade formaldehyde into carbon dioxide through a serious of reactions. At the same time, we add color reaction indicator system to achieve dynamic interaction with the user and light-controlled cracking system to ensure biosecurity.
The core part is the formaldehyde degradation system. In this system, four kinds of different protein works together. And these protein gradually degrades formaldehyde into carbon dioxide.
In order to give users the convince of keeping track of what our bacteria are doing, we add the color reaction indicator system using different color reaction to show the concentration of Indoor formaldehyde.
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.
After formaldehyde degradation, we hope that the bacteria can be effectively degraded without causing bacterial leakage and secondary pollution. In order to achieve this goal, we designed the light-dependent controlled lysis system. With this system, bacteria can express lysis genes under light conditions, cell death.
Formaldehyde reaction of formic acid need Glutathione-dependent Formaldehyde-activating Enzyme (GFA), Glutathione-dependent formaldehyde dehydrogenase (GS-FDH) and S-formylglut-athione hydrolase (FGH) which come from Paracoccus denitrificans (p. denitrificans) catalyst. GFA can act as a glutathione (GSH) carrier to catalyze the reaction of GSH and CH2O to form s-hydroxy-methylglutathione (HMG)[1], GS-FDH can catalyze the oxidation of HMG dehydrogenation to form s-formylglutathione, and FGH can regenerate GSH [2].
Figure 1. Oxidative dehydrogenation of formaldehyde
Formate dehydrogenase (LBFDH) from Lactobacillus buchneri (L. buchneri) can degrade formate into CO2.
Figure 2. Oxidative dehydrogenation of formic acid
LuxR-LuxI positive feedback amplifier:
LuxR and LuxI come from Pseudomonas aeruginosa. luxI can synthesize N-acyl homoserine lactones (N-AHL) by using fatty acyl-acyl carrier protein and s-adenosyl methionine as the substrate. And LxuR could specifically recognize N-AHL and then bind to it, so as to control and regulate the expression of other target genes.
Figure 3. the action principle of LuxR and LuxI
When there is formaldehyde in the culture environment , PfrmR will be induced to express LxuI which could synthesize AHL. After AHL is combined with LxuR, PluxR will be induced to express more LuxR and LxuI. Finally, a positive feedback amplifier will be established.
Figure 3. LuxR-LuxI positive feedback amplifier
We glanced at the IGEM 2018 entries, found a team called BGIC-Global had constracted a Formaldehyde degradation bacterium. They introduce the gene(gfa) from p. denitrificans, which could control GFA expression into E. coli and then determinated the efficiency though growth curve.
With reference to their previous work, we imported GFA into E. coli BL21 and then screened out the correct first generation formaldehydetic strain (FDS-1) through colony PCR and SDS-PAGE electrophoresis. Though measuring formaldehyde tolerance, GFA enzyme activity, degradation rate of formaldehyde and the maximum amount of formaldehyde ,we found that the recombinant strain containing GFA had no significant improvement in the degradation capacity of formaldehyde compared with the original strain BL21.
Figure 4. FDS-1 formaldehyde degradation pathway
Through consulting relevant literature, we found there are GS-FDH and FGH in p. denitrificans which could catalytic dehydrogenation of formaldehyde together with GFA and found the gene (flhA) control GS-FDH expression and the gene(fghA) control FGH expression.
After introducing gfa, flhA and fghA into BL21 and screening the second generation of formaldehydetic strain (FDS-2) through colony PCR and SDS-PAGE electrophoresis, we found that the formaldehydetic tolerance of FDS-2 was improved to some extent compared with that of BL21 through the determination of its formaldehyde tolerance, enzyme activity, the degradation rate of formaldehyde and the maximum amount of formaldehyde that can be degraded.
Figure 5. FDS-2 formaldehyde degradation pathway
Since the accumulation of formic acid would affect the growth of the strain, in order to further optimize the degradation efficiency of FDS-2 for formaldehyde, we found that LBFDH existing in L. buchneri could degrade formate into CO2[2].By introducing the gene controlling LBFDH expression, we constructed the third generation of formaldehyde degrading strain (FDS-3), hoping to reduce the accumulation of formic acid in bacteria and improve the degradation efficiency of formate.
Figure 6. FDS-3 formaldehyde degradation pathway
In order not to impose too much expression pressure on the strain and to solve the serious problem of PfrmR leakage expression in the formaldehyde detection group, we introduced a positive feedback amplifier consisting of luxR and luxI [3] to constructed the fourth-generation formaldehyde degrading strain (FDS-4). The expression of cat induced by different concentrations of formaldehyde was tested to verify whether the amplifier could solve the problem of leakage expression, and the growth curve was measured to verify whether it could relieve the expression pressure of strains.
Figure 7. FDS-4 formaldehyde degradation pathway
To characterize the concentration of formaldehyde in the solution by the color of the solution, initially we considered using fluorescent proteins as indicators, but as the fluorescent protein requiring uv excitation it was not practical due to the limitation of the application scenarios we designed. Finally, at the suggestion of iGEM-UCAS members, we adopted the different color reactions between enzyme with substrate to achieve the purpose.
We chosen the pFrmR, an engineered formaldehyde-inducible promoter, to expresses the genes when the concentration of CHOH in the environment reach a certain level. However, when testing the sensitive concentration of the FrmR promoter, we found that for the system cultured overnight, there was no obvious difference in the presence of with or without formaldehyde. That is to say, the promoter leakage was relatively serious. In order to achieve the ideal expression effect, our color reaction indicator system together with formaldehyde degradation group designed the amplification system to connect luxI to our group and put the gene expressing the enzyme of color reaction on PUC18.
To control the genes to start or stop being expressed, we need a regulatory system. In consideration of the lactose operon has been used in light-dependent controlled lysis groups, Ara Operon is used in our pathways. The AraC protein would binds to AraBAD promoter, inhibiting its expression. At this time, only CAT gene is expressed in the pathway. And when the pFrmR turn off, without repressor protein the AraBAD promoter activate, the LacZ alpha is expressed. Then we will use different substrate reacting with catalase and beta-Gal to get different color changes.
In the whole process of the experiment, in order to optimize the expression of the indication pathway, we carried out a series of experiments including the selection of chromogenic agents ONPG and TMB, the determination of enzyme expression and chromogenic agents, the determination of growth curve and so on.
We used the part BBa_K2556333, which had been constructed by 2018 iGEM team ZJUT-China[1]. 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. In this system, under dark conditions, phosphorylated FixJ protein was transferred from YF1 protein to FixJ protein, and phosphorylated FixJ protein activated pFIXK2 promoter, then activated downstream gene expression. When the LacI protein binds to LacO, it prevents the downward expression of Lac promoter, the lysis gene is not activated and the cells grow normally. When induced by light, phosphorylation of FixJ protein was blocked and gene expression regulated by pfixk2 promoter was inhibited. Lac promoter expression was not inhibited, lysis gene expression was normal, cell died.
After the successful construction of the system, we tested the efficiency of lysis gene expression by controlling the light intensity.
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