Colibacillus engineering strain for aerobic fermentation
A technology of Escherichia coli and aerobic fermentation, applied in the field of genetic engineering and microbial fermentation
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Embodiment 1
[0043] Embodiment 1, bacterial strain construction
[0044] (a). Knockout of ptsG gene:
[0045] Bacteria: Escherichia coli MG1655
[0046] The LB medium is: peptone 10g / L, yeast powder 5g / L, NaCl 10g / L, ampicillin 100mg / L, kanamycin 50mg / L.
[0047] The ampicillin-resistant plate is an LB solid medium containing 100 mg / L ampicillin and 1.5% agar powder.
[0048] The chloramphenicol-resistant plate is an LB solid medium containing 50 mg / L chloramphenicol and 1.5% agar powder.
[0049] The SOC medium is: peptone 2g / L, yeast powder 0.5g / L, NaCl 0.0585g / L, KCl 0.0186g / L, MgCl 2 0.203g, MgSO 4 0.246g / L, glucose 20mmol / L.
[0050] (1) Cloning of homologous recombination fragments
[0051] The target gene was knocked out using the Red recombination system. Primers were designed according to the ptsG gene sequence published by Genbank:
[0052] pKD-ptsGF:
[0053] 5′-ACGTAAAAAAAGCACCCATACTCAGGAGCACTCTCAATTGTGTAGGCTGGAGCTGCTTC-3′
[0054] pKD-ptsGR:
[0055] 5′-AGCCATCTGG...
Embodiment 2
[0147] Embodiment 2, the growth curve comparison of engineering strains in LB medium
[0148] Strains: E.coli MG1655, E.coli Q8, E.coli Q9 and E.coli Q10 (Deposit number: CCTCC M209113)
[0149] Medium: LB medium: peptone 10g / L, yeast powder 5g / L, NaCl 10g / L.
[0150] Training method:
[0151]Single colonies of E.coli MG1655, E.coli Q8, E.coli Q9 and E.coli Q10 (Accession No.: CCTCC M 209113) were picked from the plate and transferred to a test tube containing 4ml LB for overnight culture. According to the inoculum amount of 1% by volume, it was transferred to a 300ml Erlenmeyer flask equipped with 50ml LB, 250 rpm, and cultured at 37°C. 1ml was sampled every 2h. Samples were placed at room temperature and centrifuged at 12,000 rpm for 2 min. Then remove the supernatant, wash the cells twice with 0.5M NaCl solution, and then resuspend the cells with 1ml of 0.5M NaCl solution. The optical density OD of the bacteria is the absorbance value at a wavelength of 600nm.
[0152...
Embodiment 3
[0153] Embodiment 3, engineering strain adds 1% glucose in LB biomass and acetate production comparison
[0154] Medium LBG: peptone 10g / L, yeast powder 5g / L, NaCl 10g / L, glucose 20g / L.
[0155] Strains: E.coli MG1655, E.coli Q8, E.coli Q9 and E.coli Q10 (Deposit number: CCTCC M209113)
[0156] (1) Culture method
[0157] (1) Preparation of seed solution: pick E.coli MG1655, E.coli Q8, E.coliQ9 and E.coli Q10 (preservation number: CCTCC M 209113) on the plate respectively with an inoculation loop and insert into 50ml LB culture chloramphenicol to a final concentration of 50mg / L in a 300ml Erlenmeyer flask based on the base, respectively, at 37°C, 250 rpm, and cultured for 12h.
[0158] (II) Shake flask fermentation: the seed solution was inserted into a 300ml Erlenmeyer flask equipped with 50ml LBG medium according to the volume ratio of 1% inoculum. Chloramphenicol was added to the medium of E.coli Q8, E.coli Q9 and E.coli Q10 (preservation number: CCTCC M209113) strains t...
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