Difference between revisions of "Part:BBa K2936013"

Revision as of 12:57, 21 October 2019

Formaldehyde degradation system

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.

Usage and Biology

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 improved the formaldehyde degradation pathway of the 2018 BGIC-Global team .
In this part, formaldehyde reaction of formic acid need Glutathione-dependent Formaldehyde-activating Enzyme (gfa), Glutathione-dependent formaldehyde dehydrogenase (flhA) and S-formylglut-athione hydrolase (fghA) 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), GS-FDH can catalyze the oxidation of HMG dehydrogenation to form s-formylglutathione, and FGH can regenerate GSH, at the mean time oxidize it to formic acid.

Characterize

We constructed this system on pet-28 and transferred the recombinant plasmid into BL21 for functional verification. Under the condition of 37 ℃, the OD value of the bacterial solution reached 0.4 after 4 hours of culture, and then the formaldehyde tolerance of the strain was determined by adding 96 pore plates according to the ratio of the bacterial solution and formaldehyde solution (gradient concentration) 1:1. According to the investigation of HP, we obtained the maximum tolerance concentration of the strain to formaldehyde was 200mg/L. Therefore, we divided the concentration of formaldehyde into 8 groups by the double method, and measured the OD value of bacteria solution after culture of BL21 and FDS-2 bacteria for 4 hours to determine their growth (the original data is shown in Figure 1 and the result is shown in Figure 2). It was found that strain BL21 did not grow at the formaldehyde concentration of 50mg/L, while strain FDS-2 stopped growing at the formaldehyde concentration of 100mg/L. Therefore, by longitudinal comparison, we found that the formaldehyde concentration tolerance of FDS-2 strain was higher than that of BL21 strain. Then we narrowed the concentration range of formaldehyde to further determine the maximum tolerance of FDS-2 strain (the result is shown in Figure 3). We found that FDS-2 strain was still growing at 52mg/L, but no longer growing at 54mg/L, so we chose to use the concentration of 52mg/L formaldehyde solution in the subsequent validation experiments. In order to verify the ability of engineered bacteria to degrade formaldehyde efficiently, we verified it from the following two aspects:
(1) measure the amount of formaldehyde degradation in the experimental group and the control group at the same time.
The strain only containing gfa gene was named FDS-1, while the strain containing complete pathway was named FDS-2. We tested FDS-1 and FDS-2 separately on plasmids, and used E. coli BL21 as the control group. We cultured the two groups of strains and the control group respectively at 37 ℃ with 52 mg/L formaldehyde for 25 minutes. After centrifugation, supernatant was taken and reacted with acetylacetone, OD600 value of supernatant was measured, standard curve was determined (the result is shown in Figure 2), and the concentration of each strain was calculated for comparison (the result is shown in Figure 4).
(2) measure the rate of formaldehyde degradation in the experimental group and the control group.
Two groups of strains and control strains were cultured under the same conditions, and then supernatants were centrifuged every 5 minutes to determine the absorbance value of acetylacetone reaction. Calculate the formaldehyde concentration according to the standard curve and compare it (the result is shown in Figure 5). 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 absorbance at the wavelength of 413 nm by spectrophotometer.

Experimental results

(1) 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.

(2)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. So we can get the following conclusion: our formaldehyde degradation system is effective.

Fig5. Degradation rate of E. coli BL21/DHα/FDS-1/FDS-2 against formaldehyde

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal EcoRI site found at 904
Illegal PstI site found at 1384
12
INCOMPATIBLE WITH RFC[12]
Illegal EcoRI site found at 904
Illegal NheI site found at 7
Illegal NheI site found at 30
Illegal PstI site found at 1384
Illegal NotI site found at 1745
21
INCOMPATIBLE WITH RFC[21]
Illegal EcoRI site found at 904
Illegal BglII site found at 1265
23
INCOMPATIBLE WITH RFC[23]
Illegal EcoRI site found at 904
Illegal PstI site found at 1384
25
INCOMPATIBLE WITH RFC[25]
Illegal EcoRI site found at 904
Illegal PstI site found at 1384
Illegal NgoMIV site found at 853
Illegal NgoMIV site found at 1049
Illegal NgoMIV site found at 1402
Illegal NgoMIV site found at 1662
Illegal NgoMIV site found at 2138
Illegal NgoMIV site found at 2249
1000
COMPATIBLE WITH RFC[1000]

Reference

[1]Alpdagtas S, Yucel S, Kapkac HA, Liu S, Binay B. Discovery of an acidic, thermostable and highly NADP(+) dependent formate dehydrogenase from Lactobacillus buchneri NRRL B-30929. Biotechnol Lett. 2018;40(7):1135-47.
[2]Bateman R, Rauh D, Shokat KM. Glutathione traps formaldehyde by formation of a bicyclo[4.4.1]undecane adduct. Org Biomol Chem. 2007;5(20):3363-7.
[3]Davies HG, Bowman C, Luby SP. Cholera – management and prevention. Journal of Infection. 2017;74:S66-S7.
[4]Garg N, Manchanda G, Kumar A. Bacterial quorum sensing: circuits and applications. Antonie Van Leeuwenhoek. 2014;105(2):289-305.
[5]Goenrich M, Bartoschek S, Hagemeier CH, Griesinger C, Vorholt JA. A glutathione-dependent formaldehyde-activating enzyme (Gfa) from Paracoccus denitrificans detected and purified via two-dimensional proton exchange NMR spectroscopy. J Biol Chem. 2002;277(5):3069-72.

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 (2)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. So we can get the following conclusion: our formaldehyde degradation system is effective.
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 ===Reference===
 [1]Alpdagtas S, Yucel S, Kapkac HA, Liu S, Binay B. Discovery of an acidic, thermostable and highly NADP(+) dependent formate dehydrogenase from Lactobacillus buchneri NRRL B-30929. Biotechnol Lett. 2018;40(7):1135-47.<br>