Escherichia coli for synthesizing 2 '-fucosyllactose and construction method and application thereof
A technology of fucosyllactose and Escherichia coli, applied in the field of metabolic engineering, can solve problems such as difficulty in realizing rapid regeneration of GTP
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Embodiment 1
[0039] Knockout of gene encoding EIICB Glc enzyme gene ptsG
[0040] (1) Preparation of E. coli MG27 competent with pCas9 plasmid
[0041] First, the pCas9 plasmid was transformed into E.coli MG27, spread on 50μg / mL Kan plate, and after culturing at 30°C for 12h, single colonies on the plate were picked and inoculated into fresh LB medium, and the final concentration was 50μg / mL. Kan antibiotics, 30°C, 220r / min overnight culture; transfer the overnight cultured bacterial solution to a 250mL conical flask containing 50mL LB medium at an inoculum volume of 1-2%, when the bacterial OD600 reaches 0.2, Add arabinose with a final concentration of 20-30mmol / L to induce pCas9 plasmid to express λ-Red recombinase; continue to culture until OD600 is 0.6-0.7, after ice bath for 20-30min, the bacteria are collected by centrifugation at 5000r / min Cells; wash the cells twice with pre-cooled sterile water, discard the supernatant, and suspend the cells with 400-500 μL of pre-cooled 10% gly...
Embodiment 2
[0045] integrated lactose permease LacY
[0046] To prepare competent E. coli MG28 with pCas9 plasmid, the steps are the same as in Example 1.
[0047] According to the lacY sequence of the lactose permease gene on the genome of E. coli MG1655, the upper and lower homology arms of the E. coli MG1655 locus treB, P tac The integrated fragment HAtreB-P composed of promoter and lacY gene tac -lacY, verify the length of the fusion fragment by DNA gel electrophoresis; then use the website (https: / / chopchop.cbu.uib.no / ) to design the N20 sequence of the treB gene, thereby constructing pTarget-treB containing the sgRNA of the treB locus plasmid.
[0048] The above-obtained donor fragment HAtreB-P tac -lacY and pTarget-treB plasmids were electro-transformed into competent E.coli MG28 with pCas9 plasmids, 800 μL of LB medium was added, cultured at 30°C and 220 r / min for 2 h, coated with Kan (50 μg / mL) ) and spectinomycin (50 μg / mL) overnight at 30°C. By picking a single colony for ...
Embodiment 3
[0050] Construction of GTP cofactor regeneration system
[0051] To prepare E.coli MG29 competent with pCas9 plasmid, the steps are the same as those in Example 1.
[0052] According to the sequence of GDP-polyphosphotransferase gene ppk2 of Pseudomonas aeruginosa ATCC 15692 and the guanosine inosine kinase gene gsk of Escherichia coli MG1655 genome published on NCBI, obtained by fusion PCR technology from Escherichia coli MG1655 Homologous arms upstream and downstream of locus nupG, constitutive promoter P tac and P J23119 The expression cassette HAnupG-P of the GTP cofactor regeneration system composed of ppk2 gene and gsk gene respectively tac -ppk2-P J23119 -gsk, verify the length of the fusion fragment by DNA gel electrophoresis; then use the website (https: / / chopchop.cbu.uib.no / ) to design the N20 sequence of the nupG gene, thereby constructing pTarget-nupG containing the sgRNA of the nupG locus plasmid.
[0053] The above-obtained gene integration fragment HAnupG-P...
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