Strain, and construction method and application thereof

A construction method and strain technology, applied in the field of strains, can solve the problems of random mutation of strains, increased production costs, side effects of product synthesis, etc.

Active Publication Date: 2019-09-13
MEIHUA BIOTECH LANGFANG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the white xanthan gum production bacteria produced by traditional mutagenesis, although the xanthan gum produced is white, but the potential unknown random mutations in the strain have certain side effects on the growth of the strain and the synthesis of products.
And use a large amount of alcohol decolorization can cause the waste of alcohol, and increase production cost

Method used

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  • Strain, and construction method and application thereof
  • Strain, and construction method and application thereof
  • Strain, and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0092] Embodiment 1: Construction of knockout plasmid pLO3-pigA:

[0093] The genome of MHZ-20000 was extracted using an extraction kit. The upstream and downstream homology arms of pigA used the MHZ-20000 genome as a template, and primers pigA-1SF / pigA-1SR and pigA-1XF / pigA–1XR and PrimeSTAR DNA polymerase ( TakaraBio, Tokyo, Japan) amplification. 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-pigA was obtained, and the recombinant plasmid was transferred into E.coli S17 ...

Embodiment 2

[0094] Embodiment 2: Construction of the knockout plasmid pLO3-vgb:

[0095] The vgb gene was derived from the E.coli S17 / pBBR-vgb preserved in our laboratory. The pBBR-vgb plasmid was extracted using an extraction kit, and the plasmid was used as a template for PCR to amplify using primers Pvgb-F / Pvgb-R and PrimeSTAR DNA polymerase. Amplify the vgb gene, detect the product by electrophoresis, and purify and recover the target gene band through the gel recovery kit to obtain the target gene vgb. The genome of MHZ-20000 was extracted using an extraction kit, and the upstream and downstream homology arms of pigA were amplified using the MHZ-20000 genome as a template, using primers pigA-2SF / pigA-2SR and pigA-2XF / pigA–2XR and PrimeSTAR DNA polymerase, respectively . Connect the upstream and downstream DNA fragments with the vgb gene by overlapping PCR, the connection method is pigA upstream homology arm-vgb gene-pigA downstream homology arm, the product is detected by electropho...

Embodiment 3

[0096] Embodiment 3: the construction of white xanthan gum mutant genetic engineering strain:

[0097] Pick a single colony of MHZ-20000 on the plate medium and inoculate it to contain Cm r 5ml of seed culture medium, cultured at 200rpm in a constant temperature shaker at 30°C for 14h, and picked a single colony of E.coli s17 / pLO3-vgb 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 MHZ-20000 and E.coli s17 / pLO3-vgb according to the ratio of 2:1, then suction filter until the pore size is placed on a filter membrane of 0.22 μm, 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°...

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Abstract

The invention relates to the field of microorganism genetic engineering, in particular to a strain, and a construction method and application thereof. Xanthomonas campestris is used an original strain, a key gene pigA of an xanthomonadins synthesis way is inactivated through a genetic engineering technique, a haemoglobin vgb gene is introduced, and a xanthan gum white mutant (the strain is authorized to the China General Microbiological Culture Collection Center for preservation with the preservation number being CGMCC No.14771) is constructed. White xanthan gum can be produced, synthesized xanthan gum basically excludes the xanthomonadins, side effects of unknown mutation to strains can be reduced, the usage quantity of ethanol can be massively reduced, resources and the production cost can be saved, and the strain has great industrial application value.

Description

technical field [0001] The invention relates to the field of microbial genetic engineering, in particular to bacterial strains, their construction methods and applications. Background technique [0002] Xanthan gum (Xanthan) is a water-soluble polysaccharide polymer produced by Xanthomonas. Widely used in food, petroleum, geology and mining, medicine, environmental protection and other industries. my country is the main production area of ​​xanthan gum. In recent years, the global demand for xanthan gum has been increasing year by year, and the output of xanthan gum in my country has also been showing an increasing trend. Global sales of xanthan gum rose from 84,000 tons in 2011 to 182,000 tons in 2015. Food processing and oil drilling and mining will still be the most important xanthan gum consumption fields at home and abroad. [0003] Xanthan gum is odorless, non-toxic, safe to eat, and has excellent properties. It is widely used in the food field. Xanthan gum needs a ...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/74C12P19/06C12R1/64
CPCC07K14/195C07K14/805C12N15/74C12P19/06
Inventor 胡炎华马挺戴晓慧黄金鑫常利斌李岩
Owner MEIHUA BIOTECH LANGFANG CO LTD
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