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Integrated vector plmo033 based on genome island and its preparation method and application

A technology for integrating vectors and genome islands, which is applied in the field of preparation of integrated vectors to achieve the effects of enriching species and increasing screening efficiency

Inactive Publication Date: 2019-05-24
SHANGHAI JIAOTONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in hyperthermophilic archaea, there are no reports on the mobility of genomic islands and the development of genetic tools using genomic islands

Method used

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  • Integrated vector plmo033 based on genome island and its preparation method and application
  • Integrated vector plmo033 based on genome island and its preparation method and application
  • Integrated vector plmo033 based on genome island and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1, the insertion of the int / att element constitutes the transitional plasmid pLMO032

[0050] PCR amplified the int / att sequence 1263bp in the genome island (such as figure 1 and 2 shown) (the amplification conditions and amplification system of the genomic island int / att sequence involved in this example are well known in the art), and the int / att base sequence is shown in SEQID NO.2. Since the 5' end of the primer has a Spe I restriction site, the PCR product and the suicide vector pLMO03 can be digested with Spe I by Spe I, and after dephosphorylation of the linearized plasmid, it can be ligated by T4 DNA Enzyme ligates the PCR product after digestion with the linearized plasmid to construct an intermediate transitional plasmid pLMO032;

[0051] Wherein, the suicide vector pLMO03 comprises an ampicillin resistance gene and its promoter, and a β-hydroxy-β-methylglutaryl coenzyme a (HMG-CoA) reductase fused with a strong promoter of glutamate dehydrogenase ...

Embodiment 2

[0052] Example 2, construction of integration vector pLMO033

[0053] Further modification on the basis of the obtained transitional plasmid, the process is as follows:

[0054] (1) Transform the transitional plasmid pLMO032, excise the non-essential gene pyrF. The pyrF gene carried in the suicide plasmid pLMO03 can be used as a negative selection marker for auxotrophic strains during the homologous recombination process, but this gene function is not needed in the integration vector, and it was removed in order to reduce the size of the vector. After analysis of the restriction enzyme sites, the two ends of the gene contain restriction sites Pst I and Sal I respectively, and the gene is removed by the action of two restriction endonucleases, leaving a transformed linearized plasmid .

[0055] (2) Construction of multiple cloning sites: In this example, a multiple cloning site MSC was synthesized, which mainly includes the following: Pst Ⅰ, Not Ⅰ, Xho Ⅰ, Sci I, Bss H Ⅱ, Bp...

Embodiment 3

[0063] Embodiment 3, implementation effect

[0064] The integration vector pLMO033 has the characteristics of a shuttle vector and can be replicated in E. coli DH5α to obtain a large number of plasmids. The obtained pLMO033 integration vector was transformed into hyperthermophilic archaea P. yayanosii A1, positive clones were screened for resistance to simvastatin, and total DNA was extracted for PCR verification. Through the analysis of the integration site of the integration vector, the integration ability was verified, and the verification results were as follows: Figure 4 shown. The clones identified as positive were continuously subcultured in a medium without resistance, and they could still be inherited stably after 20 passages. The verification results were as follows: Figure 5 shown.

[0065] In P. yayanosii A1, the integrated vector pLMO033 can be used as a gene complementation vector. The amiE gene that has fused the strong promoter of glutamate dehydrogenas...

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Abstract

The invention discloses an integrated vector pLMO033 based on a genomic island and a preparing method and application thereof. The integrated vector pLMO033 carries an int / att element. The preparing method comprises the step of constructing an intermediate transitional plasmid pLMO032 and modifying the obtained intermediate transitional plasmid pLMO032 to obtain the integrated vector pLMO033. According to the application, the integrated vector pLMO033 is applied to gene single copy anaplerosis or exogenous gene expression in P.yayanosiiA1. The integrated vector can be copied in escherichia coli, can carry out locus specificity integration on a P.yayanosiiA1 stain efficiently, can be inherited stably, and can be applied to the gene function identification study as a gene anaplerosis vector for the P.yayanosiiA1.

Description

technical field [0001] The invention relates to a preparation method and application of an integrative vector, in particular to a genomic island-based integration vector pLMO033 and its preparation method and application. Background technique [0002] Thermococcales are a class of hyperthermophilic archaea widely distributed in geothermal environments, such as hot springs, volcanoes, and deep-sea hydrothermal vents. They are located at the root of the phylogenetic tree of the 16S rRNA gene and are considered to be a good tool for studying early life phenomena and biological evolution on Earth. Material. Due to its high temperature and anaerobic growth characteristics, the development of corresponding genetic tools for molecular genetics research has been lagging behind. The existing genetic manipulation tools are mainly gene knockout tools and self-replicating shuttle vectors, which have been reported in some members of Thermococcales such as Thermococcus kodakarensis, Ther...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/74
CPCC12N15/74
Inventor 徐俊李臻肖湘
Owner SHANGHAI JIAOTONG UNIV
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