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Escherichia coli genome integration vector, genetically engineered bacterium and application of genetically engineered bacterium to xylitol production

A technology of genetically engineered bacteria and integrated vectors, applied in the fields of genetic engineering and biology, can solve the problems of time-consuming and laborious, cumbersome experimental operation, and reduced recombination efficiency, and achieve the effect of solving time-consuming and laborious, simplifying integration technology, and reducing production costs.

Inactive Publication Date: 2015-07-22
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The most commonly used homologous recombination is Red / ET recombination, but this technology needs to find a suitable recombination site for each integration, and then design different homology arms according to the integration site, which is time-consuming and laborious; and with the purpose As the length of the gene increases, its recombination efficiency will drop significantly
Site-directed integration often uses the phage integration site in Escherichia coli for integration, and it is difficult to achieve multi-copy integration
Transposase-mediated recombination needs to use transposase to process the target gene, and then perform random integration. The experimental operation is cumbersome, and multi-copy integration is not possible.
Recently, some researchers used the FRT site as the integration site to carry out multi-copy integration. This method is relatively simple, but the resistance gene cannot be deleted, which increases the metabolic burden of the cells and may cause biological safety problems.

Method used

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  • Escherichia coli genome integration vector, genetically engineered bacterium and application of genetically engineered bacterium to xylitol production
  • Escherichia coli genome integration vector, genetically engineered bacterium and application of genetically engineered bacterium to xylitol production
  • Escherichia coli genome integration vector, genetically engineered bacterium and application of genetically engineered bacterium to xylitol production

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Construction of genetically engineered bacteria with different promoters

[0045] 1. Construction of Trc promoter expression vector

[0046] Using plasmids pET-30a(+) and pTrc99a-rbs-xr6600 as templates, primers ori-kan-P1 and ori-kan-P2 were designed to amplify the replicon and kanamycin resistance gene on pET-30a(+) , design primers Ptac+XR-P1 and Ptac+XR-P2 to amplify the promoter, target gene and terminator on the plasmid pTrc99a-rbs-xr6600, digest with enzymes respectively, connect with T4 ligase after digestion, and construct a recombinant plasmid; Transform the recombinant plasmid into Escherichia coli competent cell DH5α, pick the colony grown on the kanamycin plate, extract the plasmid and sequence it, and name the correct recombinant plasmid as follows:

[0047] pTrc99a-kan-xr6600, the plasmid was transferred into strain HK401, and a genetically engineered bacterium containing the corresponding recombinant plasmid was constructed, which was named HK...

Embodiment 2

[0078] Example 2 Test of different engineering bacteria producing xylitol ability

[0079] Inoculate engineering bacteria HK412, HK422 and HK432 in 45mL of improved M9 medium (1L medium contains 4-6g Na 2 HPO 4 , 2~5g KH 2 PO 4 , 1~2g NH 4 Cl, 1~5g NaCl, 1~5mM MgSO 4 , 1~5mM CaCl 2 , 2~10g / L yeast extract), cultured at 30°C until OD600 was 0.6~1, added an appropriate amount of inducer (Trc: IPTG pBAD: arabinose) and added xylose to the fermentation broth to a final concentration of 20g / L, adding glucose to a final concentration of 10g / L, culturing at 30°C, investigating the fermentation characteristics of each engineered bacteria, the results of the investigation are as follows Figure 5 , 6 , 7 shown.

[0080] Such as Figure 5 As shown, the engineered bacteria HK412 can consume glucose and xylose within 34.5 hours after being induced by 0.1mM IPTG, produce xylitol 18.5g / L, and the production efficiency is 0.62g / L / h;

[0081] Such as Image 6 As shown, under the i...

Embodiment 3

[0083] Example 3 Construction of integration vector

[0084] Use primers CM+R6K-P1 and CM+R6K-P2 to amplify the R6K replicon and the chloramphenicol resistance gene carrying the FRT site using PKD3 as a template, and use primers IS5-P1 and IS5-P2 to amplify the insert sequence IS5, Link the IS5 sequence with the R6K replicon and chloramphenicol resistance by overlapping PCR, design primers pCDF43-P1 and pCDF43-P2, use pCDF43 as a template to amplify the promoter P43, XR and terminator, respectively digest, enzyme After cutting, connect with T4 ligase to construct a recombinant plasmid; transform the recombinant plasmid into Escherichia coli competent cell DH5α, pick the colonies grown on the streptomycin sulfate plate, extract the plasmid and sequence it, and verify the correct naming of the recombinant plasmid For: pRC43.

[0085] Wherein, the concrete sequence of primer is as follows:

[0086] CM+R6K-P1: 5'-AAAA CTGCAG AGTAGGGAACTGCCAGGCATCAA-3'

[0087] CM+R6K-P2: 5’-A...

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Abstract

The invention discloses an escherichia coli genome integration vector, a genetically engineered bacterium and an application of the genetically engineered bacterium to xylitol production. The escherichia coli genome integration vector comprises a replicon, an expression original, a target gene, a resistant gene and an integration site, wherein the integration site is an IS (insertion sequence). The genetically engineered bacterium comprises escherichia coli and the integration vector integrated in an escherichia coli genome. The IS with relatively high copy number in the escherichia coli genome is taken as the integration site for performing integration of the vector and the genome, so that an integration method is simple, the target gene integrated on the genome is genetically stable, problems of high metabolic burden, unstable separation and uncontrollable protein expression of engineered bacterium when a plasmid serves as an expression vector are solved, an existing integration technology is simplified, and problems of complexity, high time consumption and the like of multi-copy integration in the existing integration technology are solved.

Description

technical field [0001] The invention relates to the fields of genetic engineering and biotechnology, in particular to an Escherichia coli genome integration vector, genetic engineering bacteria and the application in producing xylitol. Background technique [0002] Lignocellulosic biomass is the most abundant and cheapest renewable resource on the earth. With the depletion of non-renewable resources such as fossil fuels, the development and utilization of renewable resources have gradually attracted people's attention. Using biomass resources to produce bio-based chemicals Products and fuels are current research hotspots. Lignocellulose includes cellulose, hemicellulose and lignin, among which the use of cellulose to produce fuel ethanol has been well studied and there are industrial production examples; lignin is relatively complex in composition, and research is relatively slow, and no significant progress has been made; accounting for about The study of hemifibers with 5...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N1/21C12P7/18
Inventor 吴绵斌苏卜利林建平杨立荣
Owner ZHEJIANG UNIV
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