Construction and application of escherichia coli capable of accumulating shikimic acid
A technology of Escherichia coli and recombinant Escherichia coli, which is applied in the field of genetic engineering and bioengineering, can solve problems such as difficulty in meeting large-scale production, and achieve the effect of increasing accumulation
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[0032] (3) Preparation of Escherichia coli Electrotransformation Competent Cells
[0033] 1. Pick a single colony from the plate and inoculate it into a Erlenmeyer flask containing 50 mL of LB medium, and shake it overnight at 30°C and 200 rpm.
[0034] 2. Inoculate in 50ml LB medium with 1% (v / v) inoculation amount, and cultivate to OD with shaking at 30°C and 200rpm. 600 When = 0.2, add 500 μL of 1 mol / L arabinose (final concentration of 10 mmol / L) to induce, 30 ° C, 200 rpm shaking culture to OD 600 = 0.6.
[0035] 3. Transfer the bacterial liquid to a 50 mL centrifuge tube and cool on ice for 10 min, then centrifuge at 4°C and 4000 rpm for 5 min to collect the bacterial cells, and wash the bacterial cells three times with 25 mL of cooled 10% (v / v) glycerol.
[0036] 4. Resuspend the cells with 500 μL of pre-chilled 10% (v / v) glycerol, and store 80 μL of each tube at -80°C.
[0037] (4) Electroporation of DNA fragments
[0038] 1. Put the competent cells, the upstream a...
Embodiment 1
[0044] Example 1: Construction of Escherichia coli shikimate kinase ΔaroL deletion strain SA2
[0045] Specific steps are as follows:
[0046] (1) The pCas9 plasmid was transformed into Escherichia coli HGXΔtyrP to construct strain SA1 (pCas9), and the strain SA1 (pCas9) was made competent for electrotransformation.
[0047] (2) Design specific primers according to the upstream and downstream 550bp gene sequences of aroL in the genome of the strain, and use the genome of the strain HGXΔtyrP as a template to amplify the gene aroL with aroL-UF / aroL-UR, aroL-DF / aroL-DR The gene sequence of the upstream and downstream genes is 550bp.
[0048] PCR conditions were as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15s; annealing at 60°C for 15s; extension at 72°C for 30s, 30 cycles.
[0049] (3) using fusion PCR technology to fuse the upstream and downstream 550bp gene sequences obtained in step (2) to obtain the homology arm of aroL.
[0050] (4) Designing ...
Embodiment 2
[0052] Example 2: Construction of Escherichia coli shikimate kinase (ΔaroL and ΔaroK) deletion strain SA3
[0053] Specific steps are as follows:
[0054] (1) The pCas9 plasmid was transformed into Escherichia coli SA2, the strain SA2 (pCas9) was constructed, and the strain SA2 (pCas9) was made competent for electrotransformation.
[0055] (2) Design specific primers according to the upstream and downstream 550bp gene sequences of aroK in the genome of the strain, and use the genome of the strain HGXΔtyrP as a template to amplify the gene aroK with aroK-UF / aroK-UR, aroK-DF / aroK-DR The gene sequence of the upstream and downstream genes is 550bp.
[0056] PCR conditions were as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15s; annealing at 60°C for 15s; extension at 72°C for 30s, 30 cycles.
[0057] (3) using fusion PCR technology to fuse the upstream and downstream 550bp gene sequences obtained in step (2) to obtain the homology arm of aroK.
[0058]...
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