Difference between revisions of "Part:BBa K3823011"

Latest revision as of 13:35, 12 October 2023

Sqr from C. pinatubonensis
There is a SQR (Sulfide: quinone oxidoreductase) from C. pinatubonensis.

Sulfide: quinone oxidoreductase, is an ancient flavoprotein of the disulfide oxidoreductase family that is present in nearly all domains of life. SQRs were first found in sulfide trophic bacteria, later SQR-like enzymes were found in the mitochondria of some fungi, as well as in all animal species whose genomes have been sequenced. Several SQRs have been purified and characterized by biochemical methods. They are considered to be integral monotopic membrane proteins, associating with the membrane through amphipathic helices. The monomeric molecular mass of the enzyme is around 50 kDa. The enzyme usually harbors a covalently-bound FAD cofactor in each monomer^[1,2].

References

• [1] Wakai, S., et al., Purification and characterization of sulfide : quinone oxidoreductase from an acidophilic iron-oxidizing bacterium, acidithiobacillus ferrooxidans.Bioscience Biotechnology and Biochemistry, 2007. 71(11): p. 2735-2742.
• [2]Zhang, Y. and J.H.Weiner, Characterization of the kinetics and electron paramagnetic resonance spectroscopic properties of Acidithiobacillus ferrooxidans sulfide:quinone oxidoreductase (SQR). Archives of Biochemistry and Biophysics, 2014. 564: p. 110-119.

SQR Functional Validation: SCU-China 2023

sqr gene under constitutive promoter

This part consists of sqr, aiming to reduce the amount of hydrogen sulfide. The gene in this part come from a soil bacterium that is harmless and the gene can expresse normally SQR is a membrane protein that effectively convertes diffusible H2S to indiffusible hydrogen polysulfide, which is effective in reducing hydrogen sulfide levels in the environment.

Design and Properties

Sqr gene ligated downstream of the constitutive promoter PJ23110 for constitutive expression of Sulfide:quinone oxidoreductase

In order to prove that our recombinant strain can reduce hydrogen sulfide in the environment, we mixed the strain with 100 mgS/L Na2S, and detected the hydrogen sulfide content in the environment with the change of time. The experimental group was a mixture of recombinant E. coli and Na2S, which showed a clear trend of S2- decrease and the degree of S2- reduction was significantly greater than that of the wild-type control group. It can basically prove that the recombinant strain can effectively degrade hydrogen sulfide in the environment.

Experimental approach

1. Transform the plasmids into E. coli DH5α competent cells.
2. The engineered bacteria are cultured in 15 mL LB- Kanamycin (1µl/ml) medium overnight at 37℃, 200rpm;
3. Take 3 mL culture, centrifuge it at 8000rpm for 10minutes. Discard the liquid.
4. Resuspend collected bacteria with 15 mL M9- Kanamycin (1µl/ml) medium.
5. Culture overnight at 37℃, 200rpm, until OD600 is up to 0.8.
6. Centrifuge it at 8000rpm for 10minutes. Discard the liquid.
7. Resuspend collected bacteria with 3 mL M9- Kanamycin (1µl/ml) medium.
8. Take 900 µL culture, mix it with 100 µL 100 mgS/L Na2S, dilute the sample tenfold.
9. Measure the OD670 with H2S content Assay Kit every 30 minutes.

Sequence and Features