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Evolution method of inducible promoter and application thereof
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A promoter, inducible technology, applied in the biological field, can solve the problems of high sensitivity, low inducible expression intensity, and inappropriate response threshold of inducers.
Active Publication Date: 2021-07-02
JIANGNAN UNIV
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[0005] The technical problem to be solved by the present invention is that existing inducible promoters have too low inducible expression intensity and inappropriate elicitor response threshold (sensitivity bias) in the application process. high or low)
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[0062] Add 0g / L, 1g / L, 3g / L, 5g / L, 7g / L, 10g / L maltose to the LB medium glvc The promoter strains were cultured, and after culturing at 30°C and 220r / min for 12 hours, the effect of different concentrations of maltose on the promoter P was detected by the fluorescence intensity of the cells. glvc response threshold. The result is as Figure 4 , the fluorescence response value after the addition of maltose showed that the threshold of the promoter was in the range of 0-3g / L.
[0063] Will P glvc The promoter is used for the verification of the screening method, and the Bacillus subtilis WB600 transformed into the plasmid PHT01MT is first cultivated to OD 600 About 1.0, add different concentrations of maltose and screening markers, after culturing for 6 hours, detect OD 600 , the bacterial growth is as follows image 3 , the results showed that when tetracycline was added, the e...
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Abstract
The invention discloses an evolution method of an inducible promoter and an application thereof, and belongs to the technical field of biology. According to the invention, maltose-induced promoters are mutated through error-prone PCR to obtain a promotermutantlibrary, and then high-throughput screening of mutants is carried out in a coupled cell growth manner to obtain promoter mutants with different response sensitivities and strengths; a gene for coding the fusion protein is connected behind the maltose inducible promoter, and the fusion protein can simultaneously play roles in double selection markers and quantitative report of gene expression quantity. The response threshold range of the inducible promoter obtained through evolution to inducermaltose is expanded from 0-3 g / L to 0-25 g / L, and the highest inducible expression intensity is improved by about 3.5 times compared with that of an original promoter.
Description
technical field [0001] The invention relates to a method for the evolution of an inducible promoter and its application, in particular to a method for the evolution of a maltose-inducible promoter and its application, and belongs to the field of biotechnology. Background technique [0002] The promoter is the recognition sequence of RNApolymerase, which can initiate the transcription of DNA and plays an important role in the expression of foreign proteins. Because inducible promoters have the advantages of simple operation and controllability, in recent years, people have conducted extensive research on the inducible expression of inducible promoters, and developed different inducible expression systems to achieve high-efficiency expression of heterologous proteins or The precise regulation of gene expression has been widely used in the fields of enzymeengineering, proteinengineering and metabolic engineering. For example, in Escherichia coli, the T7 expression system ca...
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