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Recombinant escherichia coli and method for applying same to produce poly(3-hydroxybutyrate-3-hydroxyvalerate) (PHBV) by utilizing single carbon source

A technology for recombining Escherichia coli and Escherichia coli, which is applied in the field of genetic engineering and microbial fermentation, can solve the problems of complex control strategies and high cost of PHBV

Inactive Publication Date: 2013-06-12
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] Aiming at the problems in the prior art that the cost of synthesizing PHBV using propionic acid is too high and the control strategy is too complicated, the object of the present invention is to provide a method for recombinant Escherichia coli and using the recombinant Escherichia coli to produce PHBV with a single carbon source

Method used

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  • Recombinant escherichia coli and method for applying same to produce poly(3-hydroxybutyrate-3-hydroxyvalerate) (PHBV) by utilizing single carbon source
  • Recombinant escherichia coli and method for applying same to produce poly(3-hydroxybutyrate-3-hydroxyvalerate) (PHBV) by utilizing single carbon source
  • Recombinant escherichia coli and method for applying same to produce poly(3-hydroxybutyrate-3-hydroxyvalerate) (PHBV) by utilizing single carbon source

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1. Strain Construction

[0031] (a). Knockout of prpC gene:

[0032] 1) Preparation of Escherichia coli DH5α Competent Cells

[0033] ①Pick a single colony of Escherichia coli and inoculate it in an LB liquid test tube, and culture it with shaking at 37°C overnight. Then inoculate the overnight culture in 30 ml of fresh LB liquid medium according to 1% transfer amount (volume ratio), and culture with vigorous shaking at 37°C, about 2h to OD 600 0.4 to 0.5.

[0034] ②Centrifuge at 4,000 rpm for 10 minutes at 4°C to collect bacteria. followed by 10 mL of ice-cold 0.1 M CaCl 2 suspended bacteria.

[0035] ③Centrifuge at 4000 rpm for 10 minutes at 4°C to collect bacteria. followed by 10 mL of ice-cold 0.1 M CaCl 2 Suspend the bacteria and place on ice for 30 minutes.

[0036] ④ Centrifuge at 4000 rpm for 10 minutes at 4°C to collect bacteria. Add 1 mL of ice-cold 0.1 M CaCl 2 -Glycerol solution to suspend the bacteria.

[0037] ⑤ Dispense 100 microliters / ...

Embodiment 2

[0204] Example 2. Recombinant Escherichia coli QW103PT utilizes xylose fermentation to produce PHBV, wherein the total sugar concentration added is 5 g / L.

[0205] (1) Strain selection: Escherichia coli QW103PT;

[0206] (2) plate culture: the bacterial classification is inoculated on the solid LB medium plate that contains the agar that is 1.5% by mass percentage and adds the ampicillin that final concentration is 50 micrograms / ml and the spectinomycin of 25 micrograms / ml, Under the condition of 25 ℃, static culture for 8 hours;

[0207] (3) Seed culture: the bacterial strain cultivated in step (2), under sterile conditions, connect 1 to 2 loops to 20 ml with an inoculation loop and add ampicillin and 25 μg / ml final concentration of 50 μg / ml Spectinomycin LB liquid medium, under the condition of 25 ℃, 150 rpm shaking culture for 8 hours, to obtain the seed solution;

[0208] (4) Expansion culture: with the inoculum size of 5% volume ratio, inoculate the seed liquid in 200mL...

Embodiment 3

[0214] Example 3. Recombinant Escherichia coli QW103PT utilizes xylose fermentation to produce PHBV, wherein the total sugar concentration added is 20 g / L.

[0215] (1) Strain selection: Escherichia coli QW103PT;

[0216] (2) plate culture: the bacterial classification is inoculated on the solid LB medium plate that contains the agar that is 2.0% by mass percentage and adds the ampicillin that final concentration is 150 micrograms / ml and the spectinomycin of 75 micrograms / ml, Under the condition of 42°C, culture statically for 16 hours;

[0217] (3) Seed culture: the bacterial strain cultivated in step (2), under sterile conditions, connect 1 to 2 loops to 100 ml with an inoculation loop and add ampicillin and 75 μg / ml final concentration of 150 μg / ml Spectinomycin LB liquid medium, under the condition of 42 ℃, 250 rpm shaking culture for 16 hours, to obtain the seed solution;

[0218] (4) Expansion cultivation: with the inoculum size of 15% volume ratio, inoculate the seed ...

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Abstract

The invention discloses recombinant escherichia coli QW102PT and an application thereof in producing poly(3-hydroxybutyrate-3-hydroxyvalerate) (PHBV) by utilizing a single carbon source. The recombinant escherichia coli can be used for efficiently utilizing the single carbon source to synthesize the PHBV, and the mole fraction of 3HV (3-hydroxyvaleric acid) is improved from 0.45 percent to 17.5 percent compared with that of recombinant bacteria DH5 alpha (pBHR68), which has obvious significance to commercial production of the PHBV. According to the invention, the problems that the cost is overhigh as propionic acid is used as an auxiliary carbon source and control strategies during the production process are excessively complicated are solved, and a new concept of synthesizing the PHBV byutilizing a simple carbon source is created.

Description

technical field [0001] The invention relates to a recombinant Escherichia coli and a method for producing biodegradable plastics thereof, in particular to a recombinant Escherichia coli producing PHBV (poly 3-hydroxybutyrate-co-3-hydroxyvalerate) and its utilization of cheap The method for producing PHBV with a single simple carbon source belongs to the fields of genetic engineering and microbial fermentation. Background technique [0002] Polyhydroxyalkanoat (PHA for short) is a functional biological polyester synthesized by microorganisms. PHA has biodegradability, biocompatibility, gas barrier property, piezoelectricity, nonlinear optical activity and other special properties caused by functional groups. The nature of PHA determines that it has many potential application prospects such as biodegradable plastics and scaffold materials for tissue engineering. Therefore, a large number of basic and application development researches have been carried out on it at home and a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12R1/19
Inventor 祁庆生陈泉王倩魏国清
Owner SHANDONG UNIV
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