A kind of recombinant Escherichia coli and its application in synthesizing 3-hydroxypropionic acid
A technology of recombining Escherichia coli and genes, applied in the field of 3-hydroxypropionic acid preparation, can solve the problems of high price, unbalanced activity of glycerol dehydratase and aldehyde dehydrogenase, complicated construction process, etc., and achieve the effect of increasing production
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Embodiment 1
[0054] Example 1: Replacement of promoters in plasmids
[0055] Strains and plasmids: E.cili BL21(DE3) was purchased from Transgen, and expression vectors pACYCDuet-1 and pCDFDuet-1 were purchased from Novozymes.
[0056] The replacement of the promoters of plasmids pACYCDuet-1 and pCDFDuet-1 is to replace the sequence of the T7 promoter on the plasmid with the sequence of the tac promoter through homologous recombination.
[0057] Take the construction of pACYCDuet-tac plasmid as an example. Cultivate the Escherichia coli strain containing plasmid pACYCDuet-1, then use the pACYCDuet-1 plasmid as a template, use mutation primers to mutate the T7 sequence on the plasmid template into tac, and then chemically transform the template digested with DpnI endonuclease into BL21( DE3) in competent cells. Then spread on the LB solid medium plate containing 50mg / L chloramphenicol, and PCR screen positive clones. Recombinant plasmids were extracted from positive clones for sequencing ...
Embodiment 2
[0067] 1. Cloning and expression of glycerol dehydratase and glycerol dehydratase activator in Klebsiella pneumoniae
[0068] (1) Bacterial strains and plasmids: Klebsiella pneumoniae (K. peneumoniae DSM 2026) was purchased from DSM Company in Germany, Escherichia coli E.coli BL21 (DE3) was purchased from Transgen Company, and the expression vector pACYCDuet-tac was the method in Example 1 preparation.
[0069] (2) The glycerol dehydratase gene (dhaB123) (dhaB1 Gene ID: 7947197; dhaB2 Gene ID: 7947198; dhaB3 Gene ID: 7947200) and the glycerol dehydratase activator gene (gdrA) (gdrA Gene ID: 6936977) were cloned by K. peneumoniae DSM2026 was used as a template, which was amplified by conventional PCR method. (the primer sequence of amplifying glycerol dehydratase gene and glycerol dehydratase activator gene is:
[0070] dhaB1-4-F-EcoR1: CCG GAATTC ATGAAAAGATCAAAACGA TTTGCAGTACT,
[0071] dhaB1-4-R-Hind III: GTT AAGCTT GATCTCCCACTGACCAAAA GCTGG)
[0072] The amplified prod...
Embodiment 3
[0107] Embodiment 3: Shake flask fermentation experiment of recombinant bacterial strain and recombinant bacterial strain implement the expression of SDS-PAGE target protein
[0108] The recombinant Escherichia coli constructed by the method in Example 2 was inoculated into LB solid medium containing 50 mg / L chloramphenicol and 50 mg / L streptomycin sulfate, and cultured at 37°C for 16 hours to obtain activated recombinant Escherichia coli.
[0109]The activated recombinant Escherichia coli was inoculated into LB medium containing 50 mg / L chloramphenicol and 50 mg / L streptomycin sulfate, and cultured at 37° C. for 10 hours to obtain seed liquid.
[0110] The seed liquid was inoculated into 50 mL of fermentation medium containing 50 mg / L chloramphenicol and 50 mg / L streptomycin sulfate according to the inoculum size of 6% volume concentration, and cultured with shaking at 37° C. and 150 rpm. When OD 600 When it reaches about 0.6, add IPTG to the fermentation broth, the final co...
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