Pseudomonas putida enolase gene cloning method and application

A technology of pseudomonas enolase and cloning method, applied in the field of genetic engineering, can solve the problems of reducing the production cost of PHA, weak tolerance and the like, and achieve the effects of improving tolerance and low price

Pending Publication Date: 2021-06-11
LIAONING UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Aiming at the problem that PHA has relatively weak tolerance in crude glycerol, the present invention clones the stress-resistant gene enolase gene from Pseudomonas putida and introduces it into the screened bacterial strain with better tolerance to construct a recombinant bacterium , to improve the tolerance of recombinant bacteria to glycerol
This is of great significance for the synthesis of PHA from glycerol, a by-product of biodiesel with low price, to solve the environmental problems caused by the excessive accumulation of by-products of biodiesel, and to reduce the production cost of PHA.

Method used

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  • Pseudomonas putida enolase gene cloning method and application
  • Pseudomonas putida enolase gene cloning method and application
  • Pseudomonas putida enolase gene cloning method and application

Examples

Experimental program
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Effect test

Embodiment 1

[0033] Embodiment 1 A kind of Pseudomonas putida enolase gene cloning method

[0034] Present embodiment parent adopts putida Pseudomonas (P.putida KT2442)

[0035] 1. Extract the total DNA of Pseudomonas putida

[0036] From the -80°C refrigerator, use an inoculation loop to dip the Pseudomonas putida bacteria solution on the medium for overnight culture, pick a single colony on the plate and culture it overnight in 10ml culture solution, and mix with 30% glycerol for 1 : 1 volume is mixed in a sterile 1.5ml cryopreservation tube, the molecular cloning method is used to extract the total DNA genome of Pseudomonas putida and a small amount of plasmid extraction.

[0037] 2. Cloning of enolase gene

[0038] The extracted total DNA was used as a template, and the upstream primer P1 and downstream primer P2 were used as specific primers to amplify the target fragment by PCR; the sequences of P1 and P2 are:

[0039] P1: GAT AAGCTT GTATGGCAAAAATCGTCG

[0040] P2: CAT CTCGAG ...

Embodiment 2

[0068] Example 2 IPTG concentration condition optimization

[0069] Spread the recombinant bacterial liquid containing the recombinant plasmid pET28a-eno on the LB plate containing Kan resistance, and screen for positive clones. Pick a single colony and inoculate it in the culture medium containing Kan and cultivate it overnight; transfer it to fresh LB medium according to the inoculation amount of 1-3%, and cultivate it to OD at 37°C 600 About 0.5, add IPTG with final concentrations of 1 mM, 0.5 mM, and 0.2 mM respectively, and incubate at 30°C for 4 hours; collect the bacteria at 8000 rpm, and wash once with water.

[0070] After the expression of the target protein was induced, each 200ml of initial culture was resuspended with 4ml of binding buffer, ultrasonically disrupted, 400W, 5s work, 5s interval, repeated 20 times; after the bacteria were broken, they were centrifuged at 10,000rpm for 20min to remove cell debris, and collected For the supernatant and the precipitate...

Embodiment 3

[0072] Embodiment 3 cultivation temperature optimization

[0073] Spread the recombinant bacterial liquid containing the recombinant plasmid pET28a-eno on the LB plate containing Kan resistance, and screen for positive clones. Pick a single colony and inoculate it in the culture medium containing Kan and cultivate it overnight; transfer it to fresh LB medium according to the inoculation amount of 1-3%, and cultivate it to OD at 37°C 600 About 0.5, add IPTG with a final concentration of 1mM, incubate at 20°C, 25°C, and 30°C for 4h; collect the bacteria at 8000rpm, and wash once with water.

[0074] After the expression of the target protein was induced, each 200ml of initial culture was resuspended with 4ml of binding buffer, ultrasonically disrupted, 400W, 5s work, 5s interval, repeated 20 times; after the bacteria were broken, they were centrifuged at 10,000rpm for 20min to remove cell debris, and collected For the supernatant and the precipitate, 40 μl of the supernatant an...

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Abstract

The invention discloses a pseudomonas putida enol enzyme gene cloning method and application. In order to solve the problem that PHA synthase is relatively weak in tolerance in crude glycerol, a primer is added from a pseudomonas putida enol enzyme genome for successful amplification to obtain a 1290bp fragment, a vector pET28a-Eno is successfully constructed through enzyme digestion connection, and after enzyme digestion and sequencing verification are correct, the vector pET28a-Eno is transformed into escherichia coli BL21 to obtain expression recombinant bacteria. The concentration and temperature of IPTG (isopropyl-beta-d-thiogalactoside) under two conditions of induced expression of recombinant bacteria are optimized, so that the Eno inclusion body successfully expresses a large amount of soluble expression. The method has important significance for synthesizing the PHA by using the biodiesel byproduct glycerol with low price, solving the environmental problem caused by excessive accumulation of the biodiesel byproduct and reducing the production cost of the PHA.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and relates to the use of PCR technology to clone the enolase gene in Pseudomonas putida, introduce it into E. The problem of relatively weak force. Background technique [0002] Polyhydroxyalkanonate (Polyhydroxyalkanonate, PHA) is a kind of granular polymer biopolyester produced by many microorganisms under the condition of sufficient carbon source and lack of nitrogen source, which can be used as carbon source and energy reserve in organisms. Due to the excellent properties of PHA, it can be used in packaging materials, pharmaceutical industry and genetic engineering. The rapid development of biodiesel produced a large number of by-products. When researchers synthesized PHA by using biodiesel by-products, they found a problem, that is, microbial strains were poorly tolerant to high concentrations of glycerol. Under these conditions, PHA cannot be synthesized, let alone directly u...

Claims

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Application Information

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IPC IPC(8): C12N15/60C12N15/70C12N1/21C12P7/62C12R1/19
CPCC12N9/88C12N15/70C12P7/625C12Y402/01011Y02E50/10
Inventor 胡风庆赵小慧吴停停
Owner LIAONING UNIVERSITY
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