Here are the new parts that we designed this year:
Modified promoter of aniline dioxygenase(BBa_K2209000)
The regulating protein coding by R gene of aniline dioxygenase can bind to the promoter which inhibiting the transcription and expression of aniline dioxygenase. The key sequences of the promoter could be random mutated which interfering the binding of regulating protein and stimulating transcription and expression of the gene. Two base of the nucleotide in promoter were mutated in this study.
Q gene of aniline dioxygenase from environmental samples(BBa_K2209001)
Q gene is the most important gene in aniline dioxygenase which had been confirmed relating with the degradation spectrum. Bacterial strain from different environment might have different degradation spectrum, compared with previous obtained strain, the combined gene (modified promoter and amplified Q gene) were expressed well.
The function of modified gene verification should be performed. In this present study, the combined plasmid was transferred to expression strain P.putida KT2440 through triphilicity. Donor strain was E.coli DH-5α, E.coli HB101（pRK2013）act as auxiliary strains and recipient bacterium was P.putida KT2440. The fuse bacterium was further using to carry out the verification experiment, it showed higher expression level and could degrade aniline analogue high-efficiency.
We also contributed to the characterization a previous part this year（BBa_K1894001）
We verified the part BBa_K1894001. Electrophoresis was carried out after cut with the restriction endonuclease, expected band was obtained. The obtained shuttle plasmid contain gene GvpA1 was cloned and transfected. Microcystis aeruginosa from TaiHu Lake were collected and treated, then cultured in laboratory as receptor cells, and transfected with the plasmid mentioned above. After cultured several days, results showed that many of the microcystis were sunk as expected, it can be seen in the figure below. This suggested that gene GvpA1 modified in the plasmid were expressed and the functions were preliminarily demonstrated. We plan to extracted the protein and verified with west blotting in further research.
Figure Modified gene in shuttle plasmid transfer to Microcystis aeruginosa collected from TaiHu Lake