Trc promoter mutation library and application thereof
A promoter and library technology, applied in the biological field, can solve problems such as inability to meet production requirements, weak strength, and limited use
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Embodiment 1
[0052] Example 1: Error-prone mutations of the Trc promoter
[0053] Trc promoter is a constitutive promoter, which is a spliced hybrid promoter of Trp and Lac. It has higher transcription efficiency than Trp and strong promoter characteristics regulated by LacI repressor protein.
[0054] Select the Trc promoter (SEQ ID NO.42) as a template, adjust the magnesium ion concentration (1mM, 2mM, 0.5mM) and manganese ion concentration (3.0mM) in the PCR reaction system through a concentration gradient, and add non-fidelity Taq DNA polymerase Increase the mutation rate of the amplification to obtain promoters of different strengths.
[0055] Green fluorescent protein (sfGFP) was used as a reporter gene to characterize the Trc promoter strength regulating its expression. In order to construct a reporter gene expression vector and facilitate the insertion of a mutant promoter, the original unmutated Trc promoter and other promoters in the promoter library were used to regulate the ...
Embodiment 2
[0056] The construction of embodiment 2 recombinant plasmids pTrc-sfgfp and mpTrc-sfgfp
[0057] Such as figure 1 As shown in (A), using pUC57-sfgfp as a template (the construction method is disclosed in the paper "Next Generation of Tn7-Based Single-Copy Insertion Elements for Use in Multi-and Pan-Drug-Resistant Strains of Acinetobacter baumannii"), with primers pTrc1 F (SEQ ID NO.43) and pTrc1 R (SEQ ID NO.44)) were amplified to obtain the PCR product of sfGFP. Plasmid pTrc99a and the above PCR product were digested with EcoR I and Hind III at the same time, and recovered using a gel recovery kit. The target gene and pTrc99a were ligated with the target sfGFP and pTrc99a by T4 DNA ligase overnight at 16°C to obtain the recombinant plasmid pTrc- sfgfp. Add 100 μL of Escherichia coli JM109 to the above ligation reaction solution, place it on ice for 30 min, heat shock at 42 °C for 90 s, add 1 mL of LB liquid medium, incubate at 37 °C for 1 h, and coat the Kana-resistant plat...
Embodiment 3
[0058] The construction of embodiment 3 genetically engineered bacteria ECTrc-sfgfp and ECmpTrc-sfgfp
[0059] Thaw Escherichia coli JM109 competent cells on ice; add 5 μL of the recombinant plasmids pTrc-sfgfp and mpTrc-sfgfp constructed in Example 2, mix well and then ice-bath for 30 min; heat shock the above mixture at 42°C for 90 seconds, then add 1 mL LB medium, and only incubate it at 37°C and 250rpm for 1h, centrifuge at 5000rpm for 3min, then pour out the supernatant, and spread the bacterial solution on the corresponding antibiotic plate; culture it upside down for 12-16 hours until a single clone appears. The transformants were picked and verified by colony PCR to obtain positive recombinant strains. Finally, it was verified by Shanghai Sangon sequencing.
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