A protein dynamic expression control system and its application in shikimic acid production
A technology of expression regulation and regulation system, applied in the field of bioengineering, can solve problems such as growth defects, and achieve the effects of reducing cell load, reducing experimental workload, and improving application prospects.
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Embodiment 1
[0049] Growth phase promoter P in Escherichia coli in embodiment 1 GPP representation of
[0050] The present invention evaluates five kinds of Escherichia coli native growth phase promoter P GPP (P rrnC , P rrnA , P rpsA , P rpsT and P rpsL ) relative activity. 5 kinds of P GPP The nucleotide sequences of the promoters are respectively shown in SEQ ID NO.1-SEQ ID NO.5. In order to determine the relative activity of the promoter, the synthetic P GPP The promoter sequence, B0034RBS sequence, and GFP sequence (SEQ ID NO.23) were sequentially inserted into the Ptet-1 plasmid to obtain p15A-P GPP -GFP plasmid.
[0051] Further, in order to reduce the experimental error, the P lac UV5 The promoter sequence (SEQ ID NO.14), the B0034RBS sequence, and the gene sequence (SEQ ID NO.24) encoding red fluorescent protein mKate2 were inserted into p15A-P GPP -GFP plasmid, obtain p15A-P GPP -GFP-P lac UV5 -mKate2. In this plasmid, the growth phase promoter P GPP Controlling...
Embodiment 2
[0053] Embodiment 2 Characterization of Degradation Label Strength
[0054] For the determination of the degradation rate of the degradation tag, the ClpP protease gene of the E. coli MG1655 strain was knocked out using conventional CRISPR / Cas9 gene editing technology to construct a protease-deficient strain. At the same time, the gene encoding the original ClpP protease was inserted into the plasmid pTet-1 plasmid to construct the inducible protease plasmid. Five different degradation tags (LAA, DAS, DAS+4, DAS+8 or GSD) were fused to the C-terminus of the green fluorescent protein GFP, and inserted into the commercial plasmid PTrcHisA to construct a reporter plasmid.
[0055] The reporter plasmid and protease plasmid were co-transformed into protease-deficient strains by heat shock transformation method, and cultured in 50 mL LB system containing 10 mM lactose. When cell density OD 600 When 0.6 was reached, ClpP protease expression was induced with 200 ng / mL anhydrotetracy...
Embodiment 3
[0056] Example 3 The establishment and evaluation of protein dynamic expression control system
[0057] Construct p15A-P GPP -GFP-SsrA, the specific steps are as follows:
[0058] (a) The vector pTet-1 (GenBank accession number: MK234848) used is an engineered vector, which synthesizes the growth phase promoter P GPP sequence, replacing the original P in the vector pTet-1 tet Promoter (SEQ ID NO.11), the obtained plasmid is named p15A-P GPP ;
[0059] (b) Fusing degradation tags SsrA of different strengths (LAA, DAS, DAS+4, DAS+8 or GSD) at the 3' end of the GFP gene, and inserting GFP-SsrA with different degradation tags SsrA into p15A-P GPP Plasmid p15A-P GPP -GFP-SsrA.
[0060] Further, in order to reduce the experimental error, the P lac UV5 Promoter sequence (SEQ ID NO.14), B0034RBS sequence, gene sequence encoding red fluorescent protein mKate2 inserted into p15A-P GPP -GFP-SsrA plasmid, obtain p15A-P GPP -GFP-SsrA-P lac UV5 -mKate2. In this plasmid, GFP is ...
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