Difference between revisions of "Part:BBa K3823014"

Revision as of 16:12, 19 October 2021

T7-SQR-J23110-SDO-AprBA-SAT

Tongji_China 2021 hope to solve the odor problem of waste food. According to the background we have researched, we found that hydrogen sulfide and ammonia are the two main odors that cause malodor in food waste, so we decided to convert these two malodor-emitting odors into non-toxic and odorless substances, so that we could solve the problem through engineered bacteria. After literature review and intense discussions, we decided to converse hydrogen sulfide into sulfate(figure 1)

Figure 1. pathway of sulfide oxidization

We choose SQR, SDO, AprBA, SAT to oxidize sulfide, using J23110 as the promoter. But the result is not satisfying, so we decided to use the stronger T7 promoter for SQR gene. Further optimization of promoter will be done in the future.

Construction

Figure 2.construct the plasmid with four genes

Figure 2.T7-SQR-J23110-SDO-AprBA-SAT

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)

Result of RT-qPCR for four genes

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.

SDS-PAGE bands of four genes regulated by the promoter of J23110

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).

SDS-PAGE bands of four genes regulated by T7 promoter

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.

SDS-PAGE bands of T7-SQR-J23110-SDO-APR-SAT

Sequence and Features