Gene-rcsB-deficient recombinant Serratia marcescens and application thereof
A Serratia marcescens and gene technology, applied in the biological field, can solve problems such as low yield and hindering the industrialization process of microbial fermentation
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Embodiment 1
[0031] Example 1: Construction of Serratia marcescens engineering strain JNB5-1ΔrcsB
[0032] Specific steps are as follows:
[0033] Using the genome of Serratia marcescens JNB5-1 as a template, RcsB-DUF / RcsB-DUR (SEQ ID NO: 3 and SEQ ID NO: 4) and RcsB-DDF / RcsB-DDR (SEQ ID NO: 5 And SEQ ID NO: 6) are primers, the DNA fragment RcsB-U (SEQ ID NO: 7) and DNA fragment RcsB-D (SEQ ID NO: 8) are obtained by PCR amplification; the aacC3 resistance gene (SEQ ID NO :9); DNA fragment RcsB-U, DNA fragment RcsB-D, and aacC3 resistance gene were sequentially connected by overlap extension PCR to obtain DNA fragment RcsB-AacC3; DNA fragment RcsB-AacC3 and Kpn I digested linear After homologous recombination, the recombinant plasmid pUTmini was ligated to obtain the recombinant plasmid pUTmini-rcsB; the recombinant plasmid pUTmini-rcsB was transformed into Escherichia coli S17-1 to obtain the transformation product 1; the transformation product 1 was spread on LB solid medium (Contains 50μg·m...
Embodiment 2
[0034] Example 2: Production of Prodigiosin
[0035] Specific steps are as follows:
[0036] With Serratia marcescens JNB5-1 as a control, a single colony of Serratia marcescens engineered strain JNB5-1ΔrcsB obtained in Example 1 was inoculated into LB liquid medium (containing 50μg·mL -1 Apramycin and 50μg·mL -1 Clindamycin), shaking culture at 37°C and 180 rpm to the early logarithmic growth phase (OD 600 =0.6) to obtain a seed liquid; the seed liquid was inoculated into an LB liquid medium with an inoculum amount of 4% (v / v), and fermented at 30° C. and 180 rpm for 24 hours to obtain a fermentation liquid.
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