A kind of arac mutant protein and application thereof
A technology of protein and expression vector, applied in the field of AraC mutant protein and its application, can solve the problems of restricting the research on the transformation of ectoine biosynthesis pathway, lack of high-throughput screening methods, etc.
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Embodiment 1
[0044] Embodiment 1, the construction of plasmid pSM01
[0045] 1. Synthesize the double-stranded DNA molecule shown in sequence 5 of the sequence listing. In the sequence 5 of the sequence listing, the 329th to 402nd nucleotides from the 5' end are P BAD Promoter, the 445th to 1164th nucleotides are gfpuV gene.
[0046] 2. Using the double-stranded DNA molecule synthesized in step 1 as a template, a primer pair consisting of gfp-for-1 and gfp-rev-1 is used for PCR amplification to obtain a PCR amplification product (P BAD -gfpuv fragment).
[0047] gfp-for-1:5`-GGC GGTACC ATCGGCGTTAAACCC-3`;
[0048] gfp-rev-1:5`-GGT GGTACC GAGCTCGAATTCATTTATTT-3`.
[0049] 3. Digest the PCR amplified product in step 1 with restriction endonuclease KpnI, and recover the digested product.
[0050] 4. Digest the pDHC29 vector with restriction endonuclease KpnI, and recover the vector backbone (about 3600bp).
[0051] 5. Ligate the digested product of step 3 with the vector backbone of s...
Embodiment 2
[0052] Embodiment 2, discovery of AraC mutant protein
[0053] 1. Construction of AraC mutant library
[0054] Using the genomic DNA of Escherichia coli DH5a (Beijing Dingguo Changsheng Biotechnology Co., Ltd. MCC001) as a template, use primers F0 (5`-TATCATATGGCTGAAGCGCAAAATGAT-3`) and R0 (5`-TTAACTCGAGTTATGACAACTTGACGGCTACAT-3`) to amplify wild-type araC After purification, the gene was double-digested with restriction endonucleases NdeI and XhoI and fragment 1 was recovered. Using the pMAL-p2x vector as a template, use primers pmal-rev-NdeI (5`-TATCATATGCTATGGTCCTTGTT-3`) and pmal-for-XhoI (5`-AATCTCGAGGAATTCGGATCCTCTAGA-3`) to amplify a partial fragment of the pMAL-p2x vector and purify Afterwards, NdeI and XhoI were used to double digest and fragment 2 was recovered. Fragment 1 and fragment 2 were ligated with T4 DNA ligase to obtain plasmid pSM02. The schematic diagram of the structure of plasmid pSM02 is shown in figure 2 .
[0055] 以质粒pSM02为模版,利用突变引物AraCl-for(5`-G...
Embodiment 3
[0071] Example 3, the application of AraCm protein (specifically binding to ectoine and being induced by it to turn on P BAD Promoter expresses downstream reporter gene)
[0072] 1. Construction of plasmid pSM03
[0073] 1. Synthesize the double-stranded DNA molecule shown in sequence 4 of the sequence listing.
[0074] 2. Using the double-stranded DNA molecule synthesized in step 1 as a template, perform PCR amplification with a primer pair composed of F0 and R0 to obtain a PCR amplification product.
[0075] F0: 5`-TAT CATATG GCTGAAGCGCAAAATGAT-3`;
[0076] R0: 5`-TTAA CTCGAG TTATGACAACTTGACGGCTACAT-3`.
[0077] 3. Digest the PCR amplification product of step 2 with restriction endonucleases NdeI and XhoI, and recover the digested product.
[0078]4. Using the pMAL-p2x vector as a template, perform PCR amplification with a primer pair composed of pmal-rev-NdeI and pmal-for-XhoI (the target sequence is the entire vector backbone except for the multi-cloning site of te...
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