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Recombinant Serratia marcescens with rcsb gene deletion and its application

A Serratia marcescens, gene technology, applied in the direction of microorganism-based methods, bacteria, peptides, etc., can solve the problems that hinder the industrialization process of microbial fermentation and low yield

Active Publication Date: 2021-11-16
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, there are still certain defects in the existing biological methods, among which the low yield is the most important defect hindering the industrialization of microbial fermentation methods

Method used

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  • Recombinant Serratia marcescens with rcsb gene deletion and its application
  • Recombinant Serratia marcescens with rcsb gene deletion and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Construction of Serratia marcescens engineering bacteria JNB5-1ΔrcsB

[0032] Specific steps are as follows:

[0033] With the genome of Serratia marcescens JNB5-1 as a template, respectively RcsB-D-U-F / RcsB-D-U-R (SEQ ID NO:3 and SEQ ID NO:4) and RcsB-D-D-F / RcsB-D-D-R (SEQ ID NO:5 and SEQ ID NO:6) as primers, obtained DNA fragment RcsB-U (SEQ ID NO:7) and DNA fragment RcsB-D (SEQ ID NO:8) by PCR amplification; synthetic aacC3 resistance gene (SEQ ID NO : 9); DNA fragment RcsB-U, DNA fragment RcsB-D and aacC3 resistance gene are connected successively by overlap extension PCR to obtain DNA fragment RcsB-AacC3; The pUTmini plasmid was ligated after homologous recombination 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·mL -1Apramycin Sulfate), cultured upside down ...

Embodiment 2

[0034] Embodiment 2: the production of prodigiosin

[0035] Specific steps are as follows:

[0036] Taking Serratia marcescens JNB5-1 as a control, a single colony of Serratia marcescens engineering bacteria JNB5-1ΔrcsB obtained in Example 1 was picked and inoculated into LB liquid medium (containing 50 μg·mL -1 Apramycin and 50 μg·mL -1 Clindamycin), cultured with shaking at 37°C and 180rpm until the early logarithmic growth phase (OD 600 =0.6), to obtain a seed solution; the seed solution was inoculated into LB liquid medium with a 4% (v / v) inoculation amount, and fermented for 24 hours at 30° C. and 180 rpm to obtain a fermentation broth.

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Abstract

The invention discloses recombinant Serratia marcescens with rcsB gene deletion and application thereof, belonging to the field of biotechnology. The invention provides a Serratia marcescens engineering bacterium JNB5-1ΔrcsB capable of high prodigiosin production, and the Serratia marcescens engineering bacterium JNB5-1ΔrcsB is obtained by knocking out Serratia marcescens JNB5-1 obtained from the gene encoding the transcriptional regulator RcsB, the Serratia marcescens engineering bacterium JNB5‑1ΔrcsB was inoculated into LB liquid medium for fermentation for 24 hours, and the production of prodigiosin in the fermentation broth could be as high as 116.48mg / L, 2.28 times that of wild-type Serratia marcescens JNB5‑1.

Description

technical field [0001] The invention relates to recombinant Serratia marcescens with rcsB gene deletion and application thereof, belonging to the field of biotechnology. Background technique [0002] Prodigiosin (PG) is a methoxypyrrole skeleton structure substance with 3 pyrrole rings, which can be produced by Serratia, Pseudomonas, Riverella (Hahella), Vibrio (Vibrio) and marine new bacteria (Zooshikella rubidus), etc. [0003] Prodigiosin can destroy the double-stranded DNA of cells with the cooperation of copper ions, and the concentration of copper ions in cancer cells is much higher than that of normal cells (the concentration of copper ions in cancer cells is generally 3.5 times that of normal cells); bleeding bacteria Red pigment exhibits the most effective DNA damage at a pH of 6.8, and cancer cells have a pH closer to 6.8 than normal cells (the pH of normal cells is around 7.4, and the pH of cancer cells is around 6.8); In addition, prodigiosin can also promote c...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12P17/16C12R1/43
CPCC07K14/24C12P17/165
Inventor 饶志明潘学玮杨套伟尤甲甲易敢峰付维来徐美娟张显邵明龙
Owner JIANGNAN UNIV
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