Part:BBa_K3823004
SAT(ATP sulfurylase) from Acidithiobacillus spp.
Brief introduction
The APS pathway consists of an APS reductase (AprBA) and an ATP sulfurylase (SAT). These enzymes are involved in the dissimilatory sulfate reduction pathway in sulfate-reducing prokaryotes. SAT utilizes ATP and sulfate to generate APS which is further converted to AMP and sulfite by AprBA.
Characterization
1.The mRNA expression level of the target gene was detected by RT-qPCR
We extracted RNA of our engineered bacteria and wild-type bacteria for RT qPCR experiment. The relative content of target mRNA in each group was calculated based on 16s gene of E.coli. The experimental results can prove that our engineering bacteria can transcribe the mRNA of the introduced gene normally. (There is no target gene in the wild type, the relative expression of engineering bacteria is very high and the difference is large)
2.The expression level of the target protein was detected by SDS-PAGE
We first tried to introduce plasmids linked with 1/2/3/4 target genes into E.coli DH5α, and detected their protein expression levels. However, due to the low expression amount and the limitation of Spectrophotometry, we did not find significant differences between the engineered strain and the wild type protein strips.
Therefore, four target genes were added with efficient T7 promoter respectively and introduced into E.coli BL21(DE3).Under the condition of IPTG induction, all proteins of strain containing target genes and wild-type strain were extracted. SDS-PAGE experiment and Coomassie brilliant blue staining showed that the expression of each target protein could be realized in E.coli BL21(DE3).
We had hoped to construct plasmids with T7 promoter for all four genes, and then test the protein expression, but the construction process was very difficult. Finally, due to the limited time, we connected the four genes, but only the SQR gene was regulated by the T7 promoter. In the staining results, only the SQR band was obvious, but it was speculated that the other proteins should be able to express normally.
Protein function verification experiment
In view of the the limiting capacity of our laboratory to detect the intermediates in the sulfide oxidation pathway, we mainly verified the function of our engineered bacteria from the oxidation level of sulfide and the generation level of sulfate.
Characterization experiment of S2- oxidation amount
We configured a series of sodium sulfide solutions with concentration gradient and tested them with detection reagents according to certain methods. The standard curve obtained is ideal. It can be considered that our detection method can accurately reflect the relative content of sulfide in the solution within this concentration range.
We put the engineered bacteria and wild-type bacteria into a certain concentration of sodium sulfide solution, take out the bacterial solution every 30 min to detect the residual sulfide concentration. The results show that our engineered bacteria can oxidize sulfide better. (because the bacteria have a certain adsorption effect on sulfide, the initial sulfur ion concentration of the two groups of added bacterial solution is lower than blank)
Reference
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 48
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1018
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 253
- 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 48
- 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 48
Illegal NgoMIV site found at 727
Illegal NgoMIV site found at 898
Illegal NgoMIV site found at 1581 - 1000COMPATIBLE WITH RFC[1000]
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