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Method for improving tolerance of Escherichia coli to flavonoids compounds

A technology of flavonoids and Escherichia coli, applied in the field of genetic engineering, can solve the problems of unfavorable production, low content, complex chemical synthesis method and the like

Active Publication Date: 2014-07-16
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the acquisition of natural flavonoids is limited by time and region, and the content in plants is low, which is not conducive to rapid research and clinical testing, and the chemical structure is very complicated, which is very complicated by chemical synthesis, which is not conducive to large-scale production

Method used

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  • Method for improving tolerance of Escherichia coli to flavonoids compounds
  • Method for improving tolerance of Escherichia coli to flavonoids compounds

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Experimental program
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Effect test

Embodiment 1

[0015] The discovery of embodiment 1FadL

[0016] Use an inoculation needle to pick a single colony on the E.coli BL21 (DE3) plate and inoculate it in 25 mL of LB medium, and activate it for 12 hours. Inoculate 4 bottles of 25mL LB medium according to the inoculum amount of 1%, place them in a shaker (200rpm) at 37°C and culture until the stable phase, and add 25μL 0.1g / L rutin or naringenin or resveratin to three of the bottles respectively Resveratrol (dissolved in DMSO), another bottle was added with 25 μL DMSO as a control, after 3 hours of culture, 8000rpm low-temperature centrifugation collected the bacteria, washed 3 times with deionized water and followed the FOCUS TM The GlobalFractionation manual extracts membrane proteins, and removes ions in protein samples with a 2-D Clean-Up kit, and finally uses a non-interfering protein concentration determination kit to measure the concentration in the sample, and finally hydrates and loads the sample at a concentration of 80...

Embodiment 2

[0017] Embodiment 2 Construction of flavonoid-resistant engineering bacteria

[0018] According to the gene sequence of fadL in NCBI, primers were designed for amplification, respectively connected to the cloning vector pMD19-T for sequencing, and after obtaining the correct transformants, they were double digested and connected to the plasmid pACYC-Duet1 to construct the recombinant plasmid pACYC-fadL. The constructed expression vector was transformed into E.coli JM109, spread on the LB medium containing chloramphenicol (yeast extract 5g / L, peptone 10g / L, NaCl10g / L, solid medium plus 20g / L agar, 121 Sterilize at ℃ for 15 minutes), pick the transformant on the transformed plate for PCR verification, obtain the correct single colony for fermentation and extract the plasmid, transfer the plasmid into E. On the LB medium, pick the transformants on the plate after transformation and carry out PCR verification. The correct single colony is the recombinant strain, that is, the E.col...

Embodiment 3

[0019] Example 3 Determination of growth curve of highly tolerant flavonoids E.coli BL21 (DE3) engineered bacteria

[0020] Pick engineering bacteria and E.coli BL21 (DE3) single colonies containing empty plasmid pACYC-Duet1 and inoculate them into M9 medium containing chloramphenicol (13.3g / L M9CA Broth, 0.2% glucose, 1mM magnesium sulfate, 0.5μg / mL thiamine-HCl) after activation for 10 h, insert 1% chloramphenicol, 1 g / L flavonoids (rutin or naringenin or resveratrol), and different concentrations of IPTG ( 0, 100, 200, 400μM) in M9 medium, cultured at 30°C 200rpm, measure OD every 1h 600 until the strain grows to a stationary phase. According to the formula μ=d x / x*d t Calculate the specific growth rate map of the strain, and then obtain the maximum specific growth rate μ max , it was found that the μ of engineering bacteria in the environment with 1g / L rutin max is 0.486, while that of the original bacterial strain is 0.24, and the growth rate is increased by 2.0 ti...

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Abstract

The invention discloses a method for improving the tolerance of Escherichia coli (E. coli) to flavonoids compounds and belongs to the field of genetic engineering. According to the method disclosed by the invention, a fact that the increase in an expression quantity of FadL can alleviate the growth of E. coli in an adverse environment where flavonoids exist, that is, the FadL protein can improve the tolerance of E. coli to flavonoids compounds is found by a proteomics approach, then through a genetic engineering technology, fadL gene from E. coli BL21 (DE3) is over-expressed in E. coli BL21 (DE3) to obtain engineering bacteria capable of tolerating the flavonoids compounds and the flavonoids compounds are exogenously added into an E. coli growth environment. Compared to the original bacteria, the engineering bacteria have the characteristic that the maximum specific growth rate is increased by 2.53 times.

Description

technical field [0001] The invention relates to a method for improving the tolerance of Escherichia coli to flavonoids, belonging to the field of genetic engineering. Background technique [0002] Flavonoids (Flavonoids) are the most diverse polyphenolic plant secondary metabolites in nature, widely present in the plant kingdom, and more than 8,000 species have been discovered so far. Flavonoids have a wide range of pharmaceutical values, and have strong antibacterial, anti-inflammatory, anti-oxidation, anti-tumor, scavenging free radicals, immune regulation, anti-cancer, anti-virus, hypoglycemic, anti-radiation, and hypolipidemic properties. learning effect. With the gradual deepening of research on the biological activity of flavonoids, there are more and more development and utilization of them, and the demand for them is also increasing. In recent years, the market of flavonoids has increased by more than 30% every year. It has broad application prospects in the field ...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N15/70C12P17/06C12P7/22C12R1/19
Inventor 陈坚周景文王奎李江华堵国成
Owner JIANGNAN UNIV
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