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Engineering strain NXdP as well as construction method and application thereof

A technology of engineering bacteria and bacteria, applied in the field of engineering bacteria NXdP and its construction, can solve the problems affecting the yield and purity of hydrogel

Active Publication Date: 2018-11-13
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, a large amount of by-products such as poly-β-hydroxybutyrate accumulates in the cells, which affects the yield and purity of the hydrogel. In this field, there is no engineering bacteria that can reduce or even completely eliminate the production of poly-β-hydroxybutyrate

Method used

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  • Engineering strain NXdP as well as construction method and application thereof
  • Engineering strain NXdP as well as construction method and application thereof
  • Engineering strain NXdP as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] Construction of key gene knockout vectors for poly-β-hydroxybutyrate synthesis pathway:

[0088] according to figure 1 As shown in the flow chart, the genome of T-3 is extracted using the extraction kit, and the upstream and downstream homology arms of the target gene are amplified using the T-3 genome as a template, respectively using primers SEQ ID NO: 1-4 and PrimeSTAR DNA polymerase . The upstream and downstream DNA fragments were connected by overlap PCR, the product was detected by electrophoresis, and the target gene band was purified and recovered by a gel extraction kit to obtain a recombinant fragment. The recombinant fragment and pLO3 plasmid were simultaneously digested with restriction enzymes SacI and XbaI, 37°C, 90min, and the digested fragment was purified and recovered by PCR using a kit, and the recovered products of the two were digested with T4DNA ligase at 16°C After connecting overnight, the recombinant plasmid pLO3-Δgene was obtained, and the re...

Embodiment 2

[0090] Construction of key gene knockout vectors for poly-β-hydroxybutyrate synthesis pathway:

[0091] according to figure 1 As shown in the flow chart, the genome of T-3 is extracted using the extraction kit, and the upstream and downstream homology arms of the target gene are amplified using the T-3 genome as a template, respectively using primers SEQ ID NO: 1-4 and PrimeSTAR DNA polymerase . The upstream and downstream DNA fragments were connected by overlap PCR, the product was detected by electrophoresis, and the target gene band was purified and recovered by a gel extraction kit to obtain a recombinant fragment. Digest the recombinant fragment and pLO3 plasmid with restriction enzymes SacI and PacI at the same time, 37°C, 90min, use the kit to purify and recover the digested fragment by PCR, and use T4DNA ligase to incubate the recovered products at 16°C After connecting overnight, the recombinant plasmid pLO3-Δgene was obtained, and the recombinant plasmid was transf...

Embodiment 3

[0093] Construction of genetically engineered strains that do not produce poly-β-hydroxybutyrate:

[0094] Pick a single colony of T-3 on TPG solid medium and inoculate it to contain Cm r 5ml of seed medium, cultivated in a constant temperature shaker at 30°C at 200rpm for 24h, and picked a single colony of E.coli s17 / pLO3-Δgene until it contained tet r In the LB liquid medium, cultured in a constant temperature shaker at 37°C at 200rpm for 8h, 5ml of each of the two strains was centrifuged at 6000rpm for 5min to collect the bacteria, and 10mmol / L MgSO 4 The solution was washed twice, centrifuged, and then washed with 200ul of MgSO 4 Resuspend the bacteria in the solution, mix T-3 and E.coli s17 / pLO3-Δgene according to the ratio of 2:1, then suction filter until the pore size is placed on a 0.22um filter membrane, and put the filter membrane with the bacteria facing up On the non-resistance plate, incubate for 12 hours in a constant temperature incubator at 30°C for conjugat...

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Abstract

The invention provides an engineering strain NXdP as well as a construction method and application thereof, belonging to the technical fields of genetic engineering and biological materials. The preservation number of the engineering strain NXdP is CGMCC 15406, the preservation location is China General Microbiological Culture Collection Center, and the preservation time is March 1, 2018. The engineering strain NXdP constructed by using the method only produces hydrogel (Xuanfu gum) during fermentation; the yield of the final pure product is 15-22 g / L, the total yield is 30-35 g / L, and the conversion rate is 48% or above. The engineering strain NXdP can effectively increase the yield of the hydrogel, and has a very high conversion rate of carbohydrate gum, thus having a broad industrial application prospect.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and biological materials, and specifically relates to an engineering bacterium NXdP and its construction method and application. Background technique [0002] Sphingomonas sp. is a new genus proposed based on the characteristics of 16s rRNA sequence, respiratory quinone species and cell polar lipid pattern. The important feature that distinguishes it from other Gram-negative bacteria is that its cell membrane contains glycosphingolipids, but no lipopolysaccharide. Sphingomonas is widely distributed in water, soil and air. At present, research on Sphingomonas is mainly focused on the ability of this genus to degrade refractory organic pollutants. For example, diphenylfuran can be used by Sphingomonas sp.RW1 as the only carbon source; it has the ability to synthesize β-carrot The ability of protein; it has the ability to synthesize a class of acidic capsular polysaccharides. These caps...

Claims

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Application Information

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IPC IPC(8): C12N15/74C12N15/70C12N15/09C12P19/04
CPCC12N15/09C12N15/70C12N15/74C12P19/04
Inventor 马挺史忠李国强吴萌萌田学峰沈雅琪
Owner NANKAI UNIV
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