Team:ZJUT-China/Improve
Human Practices
Improvement
The purpose of our project is to build engineered bacteria to degrade formaldehyde efficiently, so as to reduce the formaldehyde concentration in the air of newly decorated houses. In order to realize the degradation of formaldehyde by engineered bacteria, we found the part BBa_K2728004 submitted by BGIC-Global team in 2018 iGEM parts deposit. In page of this part, we found that according to the control experiment of iGEM18_BGIC-Global, it could not well indicate that the strain containing gfa gene had higher degradation efficiency than the strain without gfa gene. Moreover, through our verification experiment, it was found that the strain only containing gfa gene had no ability to degrade formaldehyde.
Therefore, in this year, we improved the formaldehyde degradation pathway in the strain and submitted BBa_K2936013 as an improvement on BBa_K2728004.The design of this part is showed in Figure 1.
Our improvements include two aspects :(1) we improved the original basic part into a composite part, which later iGEM team could use directly on plasmids and genomes; (2) improved the degradation efficiency of formaldehyde. We verified the formaldehyde degradation efficiency of this pathway from two aspects. One is to measure the amount of formaldehyde degradation in the experimental group and the control group at the same time, the other is to measure the rate of formaldehyde degradation in the experimental group and the control group. The method we used was based on acetylacetone reaction. This is the principle of this method is that formaldehyde reacts with acetylacetone in the buffer solution of acetic acid ammonium acetate with pH = 6, and rapidly generates stable yellow compound in boiling water bath. Finally measure its OD value at the wavelength of 413nm by spectrophotometer. The functional comparison of original part and improved part is shown in Figure 2.
We tested BBa_K2936013 and BBa_K2728004 separately on plasmids, and used E. coli BL21 as the control group. The first step was to determine the amount of degradation. The strain only containing gfa gene was named FDS-1, while the strain containing complete pathway was named FDS-2. We cultured the two groups of strains and the control group respectively at 37 ℃ with 50 mg/L formaldehyde for 25 minutes. After centrifugation, supernatant was taken and reacted with acetylacetone, OD value of supernatant was measured, standard curve was determined (the result is shown in Figure 3), and the concentration of each strain was calculated for comparison (the result is shown in Figure 4).
From the two charts, it shows that after 25 minutes, the residual formaldehyde in FDS-2 bacterial solution was significantly less than that in FDS-1 bacterial solution. 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.
Then, we did another test. Two groups of strains and control strains were cultured under the same conditions, and then supernatants were centrifuged every 5 minutes to determine the OD value of acetylacetone reaction. Calculate the formaldehyde concentration according to the standard curve and compare it (the result is shown in Figure 5).
We found that the formaldehyde degradation rate of FDS-2 strain was significantly higher than that of FDS-1 and three control groups, 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 25 minutes, the rate curve showed that FDS-2 strain had completely degraded formaldehyde, while FDS-1 strain only degraded formaldehyde to 15mg/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. At the same time, by comparing the rate curves of E. coli DH5α and E. coli BL21, it shows that the degradation rate of E. coli BL21 is higher than that of E. coli DH5α, so we chose E. coli BL21 strain as the chassis. Through our improvement, the ability of the strain to degrade formaldehyde has been greatly improved, so we believe that the above improvement is of great significance.
