Construction and application method of a high-yielding n-acetylglucosamine Escherichia coli
A technology of Escherichia coli and acetamido, applied in the field of genetic engineering, can solve a large number of problems and achieve the effect of enhancing gene expression
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Embodiment 1
[0032] This embodiment is Escherichia coli as a host nagABE Knockout of gene clusters, the specific operation is as follows:
[0033] 1) According to the sequence of Escherichia coli BL21 (DE3) genome (Genbank No.CP001509) nagABE Gene cluster and its upstream and downstream sequences, designed primers: upstream primer nag-S1:
[0034] CTATCTGAGCTTGTCCGCCTGGTGTCATACTTTTCTCTAGTGTAGGCTGGAGCTGCTTC (shown in SEQ ID NO.1) and downstream primer nag-A1:
[0035] TGCGACGCTCAAGCGTCGCATCAGGCATAAAGCAGATTATGGGAATTAGCCATGGTCC (shown in SEQ ID NO. 2).
[0036] Using primers nag-S1 and nag-A1, and using plasmid pKD3 (GenBank: AY048742.1) as a template, the DNA fragment was amplified by conventional PCR and purified for future use.
[0037] 2) The above fragment was electrotransformed into Escherichia coli BL21 (DE3) competent cells containing the recombinant plasmid pKD46 and induced by adding arabinose, and the Red homologous recombination technology was used to knock out the nagABE g...
Embodiment 2
[0039] This embodiment is the Escherichia coli host manXYZ Knockout of gene clusters, the specific operation is as follows:
[0040] 1) According to the sequence of Escherichia coli BL21 (DE3) genome (Genbank No.CP001509) manXYZ Gene cluster and its upstream and downstream sequences, designed primers: upstream primer man-S1:
[0041] ACGTTGAGGTGTTAACGATAATAAAGGAGGTAGCAAGTGGTGTAGGCTGGAGCTGCTTC (shown in SEQ ID NO.3) and downstream primer man-A1:
[0042] AACGGGGCCAAAAGGCCCCGGTAGTGTACAACAGTCTTAATGGGAATTAGCCATGGTCC (shown in SEQ ID NO.4); using primers man-S1 and man-A1, using plasmid pKD4 (GenBank: AY048743.1) as a template, the DNA fragment was amplified by PCR and purified for later use.
[0043] 2) Electrotransform the above fragment into E. coli BL21(DE3) / ΔnagABE::Cm competent cells containing recombinant plasmid pKD46 and induced by adding arabinose, and use Red homologous recombination technology to knock out BL21(DE3) / ΔnagABE: : in Cm manXYZ Gene clusters, screened...
Embodiment 3
[0045] This example is to eliminate the resistance gene in the strain BL21(DE3) / ΔnagABE::Cm / ΔmanXYZ::Kan obtained in Example 2. The specific operation is as follows:
[0046] 1) Prepare BL21(DE3) / ΔnagABE::Cm / ΔmanXYZ::Kan competent cells, transform with pCP20 plasmid, and screen the correct transformants on the 30-degree ampicillin, kanamycin and chloramphenicol three-antibody plate.
[0047] 2) Streak the above transformants on an anti-antibody LB plate and culture at 42°C until a single colony appears.
[0048] 3) Pick a single colony and connect it to the LB plate without anti-antibody, and the double-antibody plate of kanamycin and chloramphenicol, and select the single colony that cannot grow on the double-antibody plate to eliminate kanamycin and chloramphenicol The antibiotic-resistant strain BL21(DE3) / ΔnagABE / ΔmanXYZ.
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