We looked up data in genebank and entrusted company to synthesize our plasmid . We will put the gene of this protein into engineering strain in order to regulate the rate at which engineered bacteria produce fatty acids by controling inducer's content.
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We designed a protein that binds downstream of the RNA polymerase binding site on the DNA.Thereby hindering the movement of RNA polymerase on DNA and preventing the transcription process of the organism At the same time, this protein can also bind to the inducer, causing the protein to fall off the DNA.The transcription process can be carried out normally.
We commissioned the company to help synthesize the plasmid backbone and the corresponding transcriptional regulators, and then we used enzymes to link the transcriptional regulators to the plasmid backbone.
- First, we need to integrate the nucleotide sequence of the target protein into the plasmid backbone.
- Then, we imported the resulting plasmid into the top 10 strain.
- Next，We added the inducer to the strain culture to see if there is fluorescence.
- What's more,we knocked out the genes associated with the metabolism of the inducer in the top 10 strain to engineer the strain.
- Finally,we quantitatively add the inducer to the strain culture solution to quantitatively measure the effect of the inducer concentration on the expression of transcriptional regulatory factors.
Georges Martin,Andreas Möglich,Walter Keller,Sylvie Doublié. Biochemical and Structural Insights into Substrate Binding and Catalytic Mechanism of Mammalian Poly(A) Polymerase[J]. Journal of Molecular Biology,2004,341(4).