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Recombinant bacillus coli and method for producing PHB by using biomass material and the same

A technology for recombining Escherichia coli and biomass raw materials, which is applied in the fields of genetic engineering and microbial fermentation, can solve the problems of high production cost of PHB and low utilization rate of biomass raw materials, and achieve low utilization rate, shorten fermentation cycle, and reduce production costs. Reduced effect

Inactive Publication Date: 2009-03-25
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Aiming at the problems of high production cost of PHB and low utilization rate of biomass raw materials in the prior art, the purpose of the present invention is to provide a method for producing PHB by recombinant Escherichia coli and using recombinant Escherichia coli to ferment cheap biomass raw materials

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1. Strain Construction

[0032] (a). Knockout of ptsG gene:

[0033] I) Cloning of homologous recombination fragments

[0034] Design primers P1 and P2 to plasmid pDEST TM 10 (purchased from invitrogen) was used as a template, and a recombinant fragment with chloramphenicol resistance was obtained by PCR (polymerase chain reaction) in vitro amplification.

[0035] Among them, the sequences of P1 and P2 primers are:

[0036] P1: 5′-ACGTAAAAAAAGCACCCATACTCAGGAGCACTCTCAATTGTGTAGGCTGGAGCTGCTTC-3′

[0037] P2: 5′-AGCCATCTGGCTGCCTTAGTCTCCCCAACGTCTTACGGAATGGGAATTAGCCATGGTCC-3′

[0038] The PCR reaction system is as follows: (primer concentration is 20 micromole / liter)

[0039] 5 microliters of 10× buffer;

[0040] 25mmol / LMgCl2 4 microliters;

[0041] 1 microliter of 10mmol / L four kinds of dNTP mixture;

[0042] 1 microliter each of upstream and downstream primers;

[0043] TaqDNA polymerase 0.5 microliters;

[0044] Template DNA 1 microliter, add water to ma...

Embodiment 2

[0084] Example 2. Recombinant Escherichia coli Q3 produced PHB by fermenting biomass raw materials, wherein the hydrolyzate of biomass raw materials was added to make the total sugar concentration in the culture medium 5 g / L.

[0085] (1) Strain selection: Escherichia coli Q3CCTCC NO: M 208105;

[0086] (2) Plate culture: Inoculate the strain on a solid LB medium plate containing 1.6% agar by mass percentage and adding ampicillin with a final concentration of 50 μg / ml, and culture it statically for 8 hours at 25°C ;

[0087] (3) Seed culture: the bacterial strain cultivated in step (2), under sterile conditions, connect 1 to 2 loops with an inoculation loop in 20 mL of LB medium with ampicillin at a final concentration of 50 μg / ml, 25 Under the condition of ℃, shake culture at 150 rpm for 8 hours to obtain seed solution;

[0088] (4) Expansion cultivation: with the inoculum size of 5% volume ratio, inoculate the seed solution in 200 milliliters and add the LB culture medium ...

Embodiment 3

[0094] Example 3. Recombinant Escherichia coli Q3 produced PHB by fermenting biomass raw materials, wherein hydrolyzate of biomass raw materials was added to make the total sugar concentration in the culture medium 20 g / L.

[0095] (1) Strain selection: Escherichia coli Q3 CCTCC NO: M 208105;

[0096] (2) Plate culture: inoculate the bacteria on a solid LB medium plate containing 1.6% agar by mass percentage and adding ampicillin with a final concentration of 150 μg / ml, and culture it statically for 16 hours at 42°C ;

[0097] (3) Seed culture: the bacterial strain cultivated in step (2), under aseptic conditions, inoculate 1 to 2 loops into 100 mL of LB medium with ampicillin at a final concentration of 150 μg / mL, 42 Under the condition of ℃, shake culture at 250 rpm for 16 hours to obtain seed solution;

[0098] (4) Expansion culture: with the inoculum amount of 15% volume ratio, inoculate the seed liquid in 1000mL and add ampicillin LB medium with a final concentration of...

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Abstract

The invention discloses a recomposed Escherichia coli and a method for producing PHB by using cheap biomass raw materials fermented by the recomposed Escherichia coli. The invention knocks out ptsG gene of the Escherichia coli MG1655 by homologous recombination technology, the Escherichia coli Q3 is obtained by injecting phbCAB gene cluster from alcaligenes eutrophus, and the strain is collected in China Center for Type Culture Collection on July 9, 2008, with the collection number of CCTCC NO of M 208105. The Escherichia coli Q3 can compose PHB by effective utilization of agricultural biomass raw material hydrolyzate, and a maximum concentration of the strains of 8.88g / L is reached 24 hours after the fermentation; PHB reaches a maximum concentration of 6.42g / L and the PHB content in the cell reaches 72.3 percent 20 hours after the fermentation. Under the same fermentation condition, by the technology, the output of PHB equal to that obtained through the utilization of glucose to produce PHB without reforming Escherichia coli can be obtained, and the production cost is far less than that by the simple utilization of glucose, thus having great practical value in the actual application.

Description

technical field [0001] The invention relates to a recombinant Escherichia coli and its method for producing biodegradable plastic polyhydroxybutyrate (PHB), in particular to a recombinant Escherichia coli producing PHB and its use of recombinant Escherichia coli to ferment cheap biomass raw materials to produce PHB The method belongs to the field 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 abroad. [000...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P7/62C12R1/19
Inventor 祁庆生陈泉李瑞
Owner SHANDONG UNIV
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