Contribution for iBowu_China
This year, ibowu_China concentrated on the severe environmental problem caused by pyrethroid, especially the accumulation of pyrethroid in aquatic ecosystems, which shows great toxicity to beneficial insects and human beings. Many enzymes have been reported to have function in degrading pyrethroid, we searched in previous iGEM projects and found 2022 iGEM team Warwick presented a project, namely Pyre-Pyrethroid Remediation, involved the design of an integrated system to test and degrade build-up of λ-cyhalothrin (a type of pyrethroid) in the environment. The enzyme they used is called Pyre1. According to this, we asked the company to synthesis Pyre1 onto pET28a(+) plasmid. We then transfected the plasmids into E.coli BL21(DE3) strains and used IPTG as induction. The preliminary expression conditions were set as follows: 1) induction for 20 h under 16℃; 2) induction for 4 h under 37℃. However, we did not observe the expression band of Pyre1. We wondered whether this is due to the small amount of protein expression, which is not enough for detection in SDS-PAGE assays, so we asked for help and underwent a western blot assay, which also provided a negative result.
In the figure, the left panels are SDS-Page results obtained by us. The right panels are Western Blot results obtained by our external help (Corresponding SDS-Page runs weren’t shown). 16C-1 means the bacteria was cultured at 16 degree Celsius and the supernatant was used for the test. 16C-2 means the 16 degree Celsius culture and the precipitation was used. 37C-1 means the bacteria was cultured at 37 degree Celsius and the supernatant was used for the test. 37C-2 means the 37 degree Celsius culture and the precipitation was used. To solve this problem, we then carefully reviewed the whole project in 2022 and found the key issue lies in the promoter and RBS part. The sequence Warwick uploaded contains its own promoter and RBS, so the original plasmids we constructed destroyed the previous promoter and RBS on pET28a(+), which may lead to the incompatibility for E.coli BL21(DE3). So we divided up the whole sequence and isolated Pyre to construct a new plasmid named pET28a(+)-Pyre, which finally showed the expression band of Pyre protein, which indicating a contribution for the existing part.
