A recombinant strain producing shikimic acid and its preparation method and application
A technology for recombinant Escherichia coli and strains, applied in the field of recombinant strains producing shikimic acid and its preparation, can solve the problems of unsatisfactory yield and unfavorable industrial production
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Embodiment 1
[0053] Construction of targeting plasmid pTKIP-GBAE
[0054] (1) All required genes were amplified using the chromosome of strain BW25113 as a template. The gene aroG was amplified with primers G1 (SEQ ID NO: 1) and G2 (SEQ ID NO: 2), and the PCR amplification conditions were as follows: PCR system (20 μL): template 10 ng, 10 μM primers 1 μL each, 2 × pfu enzyme P CR reaction mixture 10μL, add ddH 2 0 to 20 μL. Amplification program: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 68°C for 30 s (minus 0.5°C for each cycle), extension at 72°C for 1 min / kb, a total of 35 cycles; extension at 72°C for 10 min.
[0055] The amplified gene aroG and plasmid pETDuet-1 were digested with restriction endonuclease BamH I and ligated to obtain plasmid pETDuet-G; amplified with primers B1 (SEQ ID NO: 3) and B2 (SEQ ID NO: 4) The gene aroB was increased, and the gene aroB and the plasmid pETDuet-1 were digested with restriction enzymes BamH I and Hind III ...
Embodiment 2
[0059] Construction of targeting plasmid pTKIP-glk-galP
[0060] (1) Using the bacterial strain BW25113 as a template, use primers glk1 (SEQ ID NO: 15) and glk2 (SEQ ID NO: 16) to amplify the gene glk fragment, the amplification conditions are the same as above, and use restriction The plasmid pETDuet-glk was obtained after digestion with endonucleases EcoR I and PstI; the gene galP fragment was amplified with primers galP1 (SEQ ID NO: 17) and galP2 (SEQ ID NO: 18), and the amplification conditions were the same as those described above. It was digested with the plasmid pETDuet-glk with restriction endonucleases Bgl II and Xho I and ligated to obtain the plasmid pETDuet-glk-galP;
[0061] (2) The genes glk and galP on the plasmid pETDuet-glk-galP together with the respective rbs sequences and the T7 promoter terminator are used with primers Duet-F (SEQ ID NO: 13) and Duet-R (SEQ ID NO: 14) Amplified, the amplification conditions are the same as above, digested with restrictio...
Embodiment 3
[0063] Construction of targeting plasmid pTKIP-ppsA
[0064] (1) Using the bacterial strain BW25113 as a template, the gene ppsA fragment was amplified with primers ppsA1 (SEQ ID NO: 19) and ppsA2 (SEQ ID NO: 20). The plasmid pETDuet-ppsA was obtained after digestion with endonucleases EcoR I and Xho I;
[0065] (2) The gene ppsA on the plasmid pETDuet-ppsA together with its rbs sequence and T7 promoter terminator were amplified with primers Duet-F (SEQ ID NO: 13) and Duet-R (SEQ ID NO: 14). The multiplication conditions are the same as above, and the restriction endonucleases Apa I and Nhe I are digested and connected to the plasmid pTKIP-cat to obtain the plasmid pTKIP-ppsA, whose structure is as follows: image 3 shown.
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