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A large-scale gene recombination method and its application in the production of bio-based chemicals

A gene recombination and genome technology, applied in the field of molecular genetics, can solve the problem of low success rate and achieve the effect of high success rate, simple process and easy operation

Active Publication Date: 2016-03-30
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this method is simple and does not require too high equipment requirements, it cannot be interrupted during the exchange process, so the success rate of this method is not very high

Method used

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  • A large-scale gene recombination method and its application in the production of bio-based chemicals
  • A large-scale gene recombination method and its application in the production of bio-based chemicals
  • A large-scale gene recombination method and its application in the production of bio-based chemicals

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1: the method for obtaining fusion son by transplanting yeast genome into Escherichia coli

[0041] Strain activation: Take an appropriate amount of bacterial liquid, inoculate it in LB liquid medium at 37°C, activate at 180rpm overnight.

[0042] Protoplast preparation: Escherichia coli bacterial cells, 3mLLB, 37°C 180rpm overnight, 0.5mL was transferred to 50mLLB, the conditions were the same as above, OD=0.9, harvested, 4°C, 8000rpm×5min, washed twice with 10mM Tris (pH8.0), Resuspend in 5mL Tris (0.1M, pH8.0, sucrose, 20%, w / v), add 2mg / mL lysozyme, the final concentration is 100ug / mL, 37°C, 12hrs.

[0043] Strain activation: Take an appropriate amount of bacterial liquid, inoculate it in YPD liquid medium at 30°C, activate at 180rpm overnight.

[0044] Bacterial liquid expansion culture: Take the activated bacterial liquid and transfer it to 50mLYPD according to the inoculum amount of 1%, and cultivate at 30°C and 180rpm with an OD600 to about 2.

[00...

Embodiment 2

[0056] Example 2 Transplanting the Bacillus subtilis genome to Escherichia coli (see Example 1 for specific molecular biology methods)

[0057] After Escherichia coli is prepared as protoplasts, the genome of Bacillus subtilis is extracted using the kit. Culture the prepared protoplasts with 6 mL LB (20% sucrose, w / v) medium until the cell concentration reaches 5-50×10 7 After CFU / mL, wash once with Tris-NaCl (10mM, 250mM, pH7.0) at 10°C, 4574g×10min, resuspend with 200uL 0.1MCaCl2, ice-bath for 30min, add 10ug yeast tRNA, mix gently, add to Add an equal volume of FusionBuffer (Tris20mM, NaCl500mM, MgCl220mM, PEG80008%-12%) to 400uL liquid LB containing 20uL yeast genome, shake gently for 1min, 37°C for 50min, add 10mL LB, 37°C, 180rpm, 3hrs. Spread the revived bacterial solution on the agar plate containing starch as the only carbon source to screen for advanced fusions.

Embodiment 7

[0058] Example 7: Characterization of the performance of the fermented isoprene of the fusion

[0059] Transformation: 5uLpACY-mvaE-mvaS-gppS2-iSP4 and 5uLpTrc-low plasmids were transferred into 100uL competent cells of the recombinant strain (see the article Yang, J., et al., Bio-isopreneproductionusingexogenousMVApathwayandisoprenesynthaseinEscherichiacoli.BioresourTechnol, 2012.104:p.642-7 ), ice bath for 30min, heat shock at 42°C for 90s, ice bath for 3min, add 450uLLB, revive at 37°C, 180rpm shaker for 1h. At the same time, as a positive control, take 5uLpTrc-low and 5uLpACY-mvaE-mvaS-gppS2-iSP4 plasmids and transfer them into 100uLBL21 (DE3) competent cells, and the following operations are the same as above.

[0060] Screening: Spread 100uL of revived bacterial solution on LB agar plate (Cm+Amp, 1‰), culture in 37°C incubator

[0061] Activation and expansion: Pick the white monoclonal (grown within 12 hours) from the previous step to activate vials, 3 mL LB (Cm+Amp, 1...

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Abstract

The invention discloses a large-scale genetic recombination method and application thereof in production of bio-based chemicals. According to the method, host protoplast and exogenous genome are mixed to prepare fusant, and the fusant is fermented to produce the bio-based chemicals. The method disclosed by the invention is easy to operate and has high success rate; compared with other measures, the method has low requirement on equipment; the method disclosed by the invention also can be combined with a high-throughput screening measure, and thus, the flow is relatively simple, and twofold results are realized with half the effort.

Description

technical field [0001] The invention relates to a large-scale gene recombination method and its application in the production of bio-based chemicals, belonging to the technical field of molecular genetics. Background technique [0002] Synthetic biology aims to use sequencing technology, computer modeling and simulation technology, bioengineering technology, chemical synthesis technology, etc., under the guidance of engineering ideas, to design and build new biological components, equipment and systems from scratch, or to modify existing biological components. Natural, natural biological systems are redesigned and transformed to achieve the purpose of using engineered biological systems or biological modules to process information, manipulate compounds, manufacture materials, produce energy, provide food, maintain and enhance human health, and improve the environment. The research goal of synthetic biology is very clear: to synthesize new organisms from scratch, or to engine...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/90C12N1/21C12P5/02C12R1/19C12R1/865
Inventor 咸漠杨建明任萌冯红茹
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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