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Construction method of gene engineering strain without plasmid and antibiotic resistance screening marker

A technology of resistance screening markers and genetically engineered bacteria, applied in the field of bioengineering, can solve the problems of antibiotic resistance, complexity, and low integration efficiency, and achieve the effects of stable production performance and high genetic stability

Inactive Publication Date: 2014-07-16
刘紫琦
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  • Abstract
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  • Application Information

AI Technical Summary

Problems solved by technology

Although the gene copy number of the engineered bacteria they obtained was increased due to chemically induced chromosomal evolution, the gene integration technology they used was the λInCh genome integration technology, which required 4 steps, which was relatively complicated and the integration efficiency was low, and the engineered bacteria still contained Antibiotic resistance selection marker, not suitable for industrial production

Method used

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  • Construction method of gene engineering strain without plasmid and antibiotic resistance screening marker
  • Construction method of gene engineering strain without plasmid and antibiotic resistance screening marker
  • Construction method of gene engineering strain without plasmid and antibiotic resistance screening marker

Examples

Experimental program
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Effect test

Embodiment 1

[0051] Construction of Lycopene-producing Engineering Bacteria Using Escherichia coli BW25113 as Starting Bacteria

[0052] 1) Transform the helper plasmid pAH121 expressing integrase (a gift from Professor Wanner of Purdue University, see Haldimann, A., Wanner, B.L. Journal of Bacteriology.2001, 183: 6384-6393) into E. coli E. coli BW25113 to obtain Escherichia coli E. coli BW25113 (pAH121).

[0053] 2) Hind III digestion of pB-crtEIBipi (gifted by Professor Liu Jianzhong of Sun Yat-Sen University, expression vector containing lycopene synthesis gene) and filled in, then digested with EcoR I, gel recovery of 4329bp lycopene synthesis gene crtEIBipi fragment , connected to the pP21KF3T5b vector (SEQ ID NO: 2) digested with EcoR I / BamH III to obtain pP21KF3T5b-crtEIBipi.

[0054] 3) Gene integration: transform pP21KF3T5b-crtEIBipi into Escherichia coli E.coli BW25113(pAH121): add 5 μl of pP21KF3T5b-crtEIBipi to 100 μl of E.coli BW25113(pAH121) competent cells, and place on ice...

Embodiment 2

[0064] Escherichia coli BW25113 (ΔgdhAΔaceF, P T5 -dxs) is the construction of the lycopene-producing engineering bacterium of the starting bacterium

[0065] Change Escherichia coli E.coli BW25113 in Example 1 into Escherichia coli E.coli BW25113 (ΔgdhAΔaceF, P T5 -dxs) (gifted by Professor Liu Jianzhong of Sun Yat-sen University), the lycopene synthesis gene crtEIBipi was connected to the chromosomal evolution plasmid pHKKF3T5b to obtain pHKKF3T5b-crtEIBipi, and the helper plasmid for expressing integrase was pAH69, and the other steps were the same as in Example 1. Finally, the chromosomally evolved E.coli CIChE-crtEIBipi tolerant to 8 μM triclosan was obtained, and the shaker fermentation could produce 30.5 mg / g dry lycopene.

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Abstract

The invention discloses a construction method of a gene engineering strain without plasmid and antibiotic resistance screening marker. A pXKF3T5b plasmid is used as the carrier to transform a target gene into a host cell, and then an auxiliary plasmid for expressing the integrase is removed, a kanamycin resistance gene in the integrated carrier is removed, the chromosome is induced by the triclosan to evolve and the evolved strain is fermented and screened, so as to obtain the gene engineering strain without plasmid but with high gene copy number; moreover, a screen marker of the pXKF3T5b plasmid is the fabI gene (for coding enoyl-acetyl carrier protein reductase) in Escherichia coli and the fabI gene is the triclosan resistance gene, thus the obtained gene engineering strain does not have the antibiotic resistance screening marker, furthermore can not cause the spread of the antibiotics drug-resistant bacteria in the environment and can be suitable for the industrialized production. In addition, the target gene of the gene engineering strain is integrated into the chromosome, thus the gene engineering strain can not be lost during sub cultures, the genetic stability is high, and the production performance is stable.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to a method for constructing genetically engineered bacteria without plasmids and antibiotic selection markers. Background technique [0002] The use of genetically engineered microorganisms to produce useful substances has become a new mode of substance production. At present, the plasmid expression system containing the target gene is usually used to construct engineered microorganisms. However, this plasmid expression system often has the disadvantages that the plasmid is easily lost and unstable, and the existence of the plasmid will increase the metabolic burden of the host bacteria. What's more serious is that plasmids often contain antibiotic resistance selection markers, which will seriously affect environmental pollution and lead to the proliferation of antibiotic-resistant bacteria in the environment, thereby limiting the industrialization process of genetically ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/70C12N1/21C12R1/19
Inventor 刘紫琦
Owner 刘紫琦
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