Team:ZJUT-China/Results
Human Practices
Results
Formaldehyde Degradation System
After FDS-1 (Fig. 1) was successfully constructed in E. coli BL21, we measured its formaldehyde tolerance, the degradation rate of formaldehyde, and the time taken to completely degrade formaldehyde. Then we found that it did not improve significantly compared with E. coli BL21 without FDS-1.
Therefore, the FDS-2 (Fig. 2) was constructed. When its formaldehyde tolerance, growth curve in the 50 ppm formaldehyde environment, the degradation rate of formaldehyde and the time taken to completely degrade formaldehyde was measured. We found that FDS-2 could degrade formaldehyde quickly and efficiently.
1. Gene expression and protein production
In order to detect the expression of related proteins in FDS-2 formaldehyde degradation pathway, SDS-PAGE electrophoresis experiment was carried out, and we found that all related proteins could be correctly expressed (Fig. 3).
Since gfa in FDS-1 is located on the pET-28b and is induced by the T7 promoter, its expression level is high. In order to avoid conflicts with the repression system of the optical control group, we optimized formaldehyde degradation pathway by using pJ23119 as the promoter in FDS-2. As pJ23119 was not as strong as the T7 promoter, the expression of GFA, GS-FDH and FGH can not be observed clearly in Fig. 3.
1. Formaldehyde tolerance
Based on the fact that the our formaldehyde degradation pathway could effectively degrade formaldehyde, we speculated that FDS might be able to tolerate higher concentrations of formaldehyde than BL21, and then we measured its formaldehyde tolerance.
After several measurements, we found that FDS can not tolerate higher concentrations of formaldehyde than BL21 (Fig. 4). In addition, based on this test of formaldehyde tolerance, we selected 50 ppm as the formaldehyde concentration to be used in subsequent experiments, because this concentration is very close to the highest formaldehyde concentration of 52 ppm that FDS can tolerate and is easy to calculate.
2. Growth curve
Formaldehyde tolerance experiment showed that the formaldehyde tolerance of FDS-2 was not significantly improved compared with E. coli BL21, we speculated whether the growth status of FDS in formaldehyde containing a certain concentration was better than that of E. coli BL21, so we measured the growth curve of FDS and E. coli BL21 in formaldehyde concentration of 50 ppm.
According to the experimental results, the growth of FDS-2 in 50 ppm formaldehyde concentration was no better than BL21 (Fig. 5). We believed that the introduction of the formaldehyde degradation system could not improve the tolerance of the strain to formaldehyde.
3. Degradation rate of formaldehyde
We used to measure the efficiency of the bacterial strain in degrading formaldehyde to reflect the ability of the bacterial strain in degrading formaldehyde.
After measuring the degradation rate of formaldehyde, we found that the formaldehyde degradation rate of FDS-2 strain was significantly higher than that of FDS-1 and three control groups (Fig. 6), and the degradation rate curve of FDS-2 strain was linear within 10 minutes, while the degradation curve of FDS-1 strain coincided with that of BL21 strain. At 25th minute, the rate curve showed that FDS-2 strain had completely degraded formaldehyde, while FDS-1 strain only degraded formaldehyde to 15 mg/L. The rate curve of LB control group without bacterial solution was parallel to the X-axis, which indicated that the degradation of formaldehyde in bacterial solution environment was due to the action of bacterial strains rather than the volatilization of formaldehyde itself. Therefore, it was concluded that FDS-2 could degrade formaldehyde rapidly and efficiently by increasing the degradation rate of formaldehyde.
4. Residual formaldehyde after 25 min degradation
In order to further illustrate that FDS has stronger formaldehyde degradation capacity than BL21, we chose to measure the residual formaldehyde concentration in the solution after 25 min degradation according to the results of the formaldehyde degradation rate test.
We found that after 25 minutes, the residual formaldehyde in FDS-2 bacterial solution was significantly less than that in FDS-1 bacterial solution (Fig. 7). Therefore, the following conclusion can be drawn: under the same culture time and conditions, the degradation amount of formaldehyde in FDS-2 bacterial solution was higher than that in FDS-1 bacterial solution.
5. Enzyme activity
In order to turn formaldehyde into a more environmentally friendly release of carbon dioxide, we added the LbFDH into the degradation pathway to achieve the purpose of degrading formic acid into carbon dioxide.
In order to find an appropriate enzyme activity test method, after searching for literature and patents, we found a high-throughput screening method: first, we used the developer for preliminary screening, the formulation and concentration of the developer are shown in Fig. 8，and the color rendering effect is shown in Fig. 9.
Then we used different concentrations of NADPH to measure the OD340 value, determining that the value of OD340 was independent of ammonium formate and other concentrations (Fig. 10).
Next, we added the substrate ammonium formate, NADP+, and crude enzyme solution into the VP tube, measured the OD340, and determined the appropriate concentration of the reaction system. The appropriate results for each component are shown Fig. 11. And we defined IU：The amount of enzyme required to produce 1μmol NADPH in 1min.
The concentration of different substrates (ammonium formate, NADP+) was used to measure the OD340 of Lbfdh, and the Km and Kcat values were calculated respectively (Fig. 12).
Finally, we did an enzyme assay and found that the LBFDH gene we imported had enzyme activity in host bacteria (Fig. 13).
Future improvement
In our previous study, we found that too much proteins produciton will influence bacterial growth. Therefore, in order to avoid excessive metabolism burden on the constructed bacteria caused by multiple expression of proteins including LBFDH, we decide to design and apply a positive feedback amplifier composed of luxR and luxI, to construct FDS-4 (Fig. 14). Meanwhile, the problem of the PfrmR leak expression would be solved.
For this amplifier, 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 and there are many LBFDH and CAT in the cell (Fig. 15).
The expression of cat induced by different concentrations of formaldehyde will be tested to verify whether the amplifier could solve the problem of leakage expression. The rate of formaldehyde decomposition will be measured to determine the strain on the degradation efficiency of formaldehyde to indicate if there is further promoted. And the growth curve will be measured to verify whether it could relieve the expression pressure of strains.
Color Reaction Based Indicator System
Overviews:
We tried different substrate for the color reaction. Finally 3, 3’ ,5, 5’ -Tetramethylbenzidine (TMB) and 2-Nitrophenyl beta-D-galactopyranoside (ONPG) were selected as the substrate because obvious color change was observed. We proved that catalase that encoded by CAT gene could react with 3, 3’ ,5, 5’ -Tetramethylbenzidine (TMB) to generate blue color. The ratio of bacterial culture (OD600=0.5) to TMB is designed to be 1:4 (Fig. 2). In the same way, beta-galactosidase that encoded by LacZ alpha gene could react with 2-Nitrophenyl beta-D-galactopyranoside (ONPG) to generate yellow color. The ratio of bacterial culture (OD600=0.5) to ONPG is designed to be 1:10 (Fig.3) .
We also tried to quantitatively characterize the pathway. However, it was hard to count due to the leakage of pFrmR. (Fig. 4)
Eventually we determined the relationship between the concentration of catalase and the value of A650.
The quantitative experiment of Ccatlase-A650
Results:
Analysis:
It can be seen that as the proportion of catalase getting up, the color of the reaction solution is getting darker and the value of A650 is getting larger. From the figure. 6, when the ratio of catalase at the range from 2.5E-03 to 3.0E-02 (μl/ml), the concentration of catalase ( μl / ml ) and the value of A650 can be represented by the relation A650=13.872Ccatalase+0.0224.
Meanings:
This proves that the CAT gene can be used as an reporter gene for qualitative characterization, as the characteristics they confer on E.coil expressing catalase are easily identified and measured, and they are selectable markers.
Considerations for replicating the experiments:
FrmR Promoter has a serious leak problem. However, from our quantitative experimental characterization data to BBa_k2556051, it could indicate that although pFrmR exists promoter leak problem at the early stage of culture, the FrmR promoter was able to induce by formaldehyde between 4h and 5h.
Catalase is very sensitive to TMB, and similarly beta-Gal is very sensitive to ONPG, so the ratio of bacterial culture or enzyme to substrates is very significant for getting the obvious color reaction.
In order to solve the leak of pFrmR and 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.
light-dependent controlled lysis system
In 2019, ZJUT-China team established a light-dependent controlled lysis system successfully (Fig. 1). Through this system, we could control the lysis of bacteria by changing the external environment. Under light condition, bacteria can express lysis genes, causing cell death. On the contrary, no expression under dark conditions, and cell grew normally.
In this system, under dark conditions, phosphate group of YF1 protein will be transferred to FixJ protein, and the phosphorylated FixJ protein will initiate the activation of FIXK2 promoter to drive the expression of downstream gene including eGFP and LacI. When the LacI protein was expressed, it will prevent the function of Lac promoter. As a result, the lysis gene will not be expressed, bacteria 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 was expressed and cell lysed. We used the light-controlled system and the lactose operon and negative induction system to establish this system.
Firstly, we measured the expression efficiency of the lysis gene by drawing the growth curve(Fig. 2).
Next, we choose the light-controlled system, which was established from ZJUT-China in 2017 (Fig. 3).
After we connected the light-controlled system with the lactose operon and negative induction system successfully, we conducted the following two experiments to verify the effectiveness of the light-dependent controlled lysis system.
1. Pick single colony into 5 mL fresh culture medium. During 14 hours of culturing in dark and light conditions, we plotted the corresponding growth curve (Fig. 4).
It can be obviously seen that the test tube cultured under light conditions was much clearer than that cultured under dark conditions, which meant our light-controlled system was effective. We did three repeated experiments and drew the corresponding growth curve. Under the light condition, the constructed bacteria are difficult to grow in the test tube. The OD600 value maintained at about 0.045. And under the dark condition, theOD600 value increased from 0.046 at first to 0.205 after 2 hours, the difference was 0.143. After that, we chose to culture the bacteria under dark conditions for 5 hours and then change it to light conditions. The results showed that when the strain was cultured under light conditions for the first 1 hour, the lysis efficiency of the strain was high. The OD600 value decreased from 0.277 to 0.180, and the difference was 0.097. This indicated that the transformation of strains from dark to light conditions would cause lysis of bacteria.
2. After the test tube experiment, we put the bacteria into the shaking flask to expand the culture. Single colonies were inoculated into two shaking flasks which containing ampicillin resistance. One shaking flask was cultured under light conditions while another one was cultured under dark conditions at 37 ℃ for 30 hours. Then, we used the Enzyme Labeling Instrument to measure the OD600. We obtained the following results (Fig. 5 and Fig. 6). The OD600 of bacteria decreased to 0.12. And under the dark condition, the OD600 is about 0.997. About 87.39% bacteria was lysed .
