AraC mutant protein and application thereof
A protein and protein technology, which is applied to AraC mutant protein and its application field, can solve the problems of lack of high-throughput screening methods, limited research on the transformation of tetrahydropyrimidine biosynthesis pathway, etc.
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Embodiment 1
[0044] Example 1. Construction of plasmid pSM01
[0045] 1. Synthesize the double-stranded DNA molecule shown in sequence 5 of the sequence listing. The nucleotides 329 to 402 from the 5'end of sequence 5 in the sequence listing are P BAD The promoter, nucleotides 445 to 1164 is the gfpuV gene.
[0046] 2. Using the double-stranded DNA molecule synthesized in step 1 as a template, PCR amplification is performed with a primer pair composed of gfp-for-1 and gfp-rev-1 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 GAGCTCGAATTCATTATTT-3`.
[0049] 3. Digest the PCR amplified product of step 1 with restriction enzyme KpnI, and recover the digested product.
[0050] 4. Cut the pDHC29 vector with restriction enzyme KpnI, and recover the vector backbone (about 3600bp).
[0051] 5. Connect the digested product of step 3 and the vector backbone of step 4 to obtain plasmid pSM01. The structure...
Embodiment 2
[0052] Example 2. Discovery of AraC mutant protein
[0053] 1. Construction of AraC mutation library
[0054] Using the genomic DNA of E. coli DH5a (Beijing Dingguochangsheng Biotechnology Co., Ltd. MCC001) as a template, the wild-type araC was amplified with primers F0 (5`-TATCATATGGCTGAAGCGCAAAATGAT-3`) and R0 (5`-TTAACTCGAGTTATGACAACTTGACGGCTACAT-3`) After purification, the gene was digested with restriction enzymes NdeI and XhoI and fragment 1 was recovered. Using pMAL-p2x vector as a template, use primers pmal-rev-NdeI (5`-TATCATATGCTATGGTCCTTGTT-3`) and pmal-for-XhoI (5`-AATCTCGAGGAATTCGGATCCTCTAGA-3`) to amplify partial fragments of pMAL-p2x vector and purify Then it was digested with NdeI and XhoI and fragment 2 was recovered. Fragment 1 and fragment 2 were ligated with T4 DNA ligase to obtain plasmid pSM02. The structure diagram of plasmid pSM02 is shown in figure 2 .
[0055] Using the plasmid pSM02 as a template, the mutant primers AraCl-for (5`-GCTGAAGCGCAAAATGATNNSCT...
Embodiment 3
[0071] Example 3. Application of AraCm protein (specifically binds to tetrahydropyrimidine and is induced to turn on P BAD The promoter expresses the 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, a primer pair composed of F0 and R0 is used for PCR amplification to obtain a PCR amplification product.
[0075] F0: 5`-TAT CATATG GCTGAAGCGCAAAATGAT-3`;
[0076] R0: 5`-TTAA CTCGAG TTATGACAACTTGACGGCTACAT-3`.
[0077] 3. Double-cut the PCR amplified product of step 2 with restriction enzymes NdeI and XhoI, and recover the digested product.
[0078] 4. Using the pMAL-p2x vector as a template, use the primer pair composed of pmal-rev-NdeI and pmal-for-XhoI for PCR amplification (the target sequence is the entire vector backbone except for the multiple cloning site of tens of bp, and the...
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