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Zymomonas mobilis-escherichia coli shuttle vector pSUZM1, pSUZM2 and pSUZM3 and construction method thereof

A technology of Zymomonas and Escherichia coli, which is applied in the field of genetic engineering, can solve the problem of large carrier and achieve the effect of strong stability and good application prospects

Inactive Publication Date: 2014-09-10
SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The existing commonly used vectors for Zymomonas mobilis mainly include spectral vectors pBBR1MCS series and RSF1010 series, etc., but these vectors have certain defects, among which the pBBR1MCS series has structural stability, while the vector RSF1010 series has a large vector

Method used

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  • Zymomonas mobilis-escherichia coli shuttle vector pSUZM1, pSUZM2 and pSUZM3 and construction method thereof
  • Zymomonas mobilis-escherichia coli shuttle vector pSUZM1, pSUZM2 and pSUZM3 and construction method thereof
  • Zymomonas mobilis-escherichia coli shuttle vector pSUZM1, pSUZM2 and pSUZM3 and construction method thereof

Examples

Experimental program
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Effect test

example 1

[0048] Construction of example 1 vector pSUZM1

[0049] For the construction strategy of the vector pSUZM1, see figure 1 ,details as follows:

[0050] 1) Design the upstream primer KanCF and downstream primer KanR of the screening marker kanamycin resistance gene; the upstream primer OriEF and downstream primer OriECR of the replication origin of Escherichia coli pUC18 plasmid; the upstream primer OriZCF and downstream primer of the ZM4 chromosome replication origin of Zymomonas mobilis OriZCR, see the specific primer sequence figure 2 .

[0051] 2) The DNA of Zymomonas mobilis was extracted separately by CTAB method. Take 2mL of bacterial liquid, centrifuge at 12000rpm for 2min, wash the precipitate with TE, and resuspend the bacterial cells with 1mLTE after centrifugation. Add 53 μL of 10% SDS, mix well, add 11 μL of 10 mg / mL proteinase K, mix well, and incubate at 37°C for 1 h. Add 211 μL 5mol / L NaCl, then add 146 μL CTAB / NaCl (50 g / L CTAB , 0.5 mol / L NaCl), mix well,...

example 2

[0066] Example 2 Construction of vector pSUZM2

[0067] For the construction strategy of the vector pSUZM2, see Figure 5 ,details as follows:

[0068]1) Design the upstream primer KanP1F and the downstream primer KanR of the screening marker kanamycin resistance gene; the upstream primer OriEF and the downstream primer OriEP1R of the replication origin of Escherichia coli pUC18 plasmid; the replication origin and DNA replicase gene of the ZM4 plasmid pZZM401 Upstream primer OriZP1F, downstream primer OriZP1R, specific primer sequence see Image 6 .

[0069] 2) The DNA of Zymomonas mobilis was extracted separately by CTAB method. See Example 1 for specific steps.

[0070] 3) Using the vector pBBR1MCS-2 as a template, KanP1F and KanR were used as primers to amplify kan-2; using the vector pUC18 as a template, OriEF and OriEP1R were used as primers to amplify oriE-2; using the whole genome of Zymomonas mobilis as a template, OriP1 was amplified with primers OriZP1F and OriZ...

example 3

[0082] Example 3 Construction of vector pSUZM3

[0083] For the construction strategy of the vector pSUZM3, see Figure 9 ,details as follows:

[0084] 1) Design the upstream primer KanP1F and downstream primer KanR of the screening marker Kanna resistance gene; the upstream primer OriEF and downstream primer OriEP1R of the E. coli replication origin; the upstream primer OriZP1F and downstream primer OriZP1R of the ZM4 chromosome replication origin of Z. mobilis, and the specific primer See sequence Figure 10 .

[0085] 2) The DNA of Zymomonas mobilis was extracted separately by CTAB method. See Example 1 for specific steps.

[0086] 3) Using the vector pBBR1MCS-2 as a template, KanP2F and KanR were used as primers to amplify the kan-3 fragment; using the vector pUC18 as a template, OriEF and OriEP2R were used as primers to amplify the oriE-3 fragment; using the whole genome of Zymomonas mobilis as As a template, use OriZP2F and OriZP2R as primers to amplify the oriP2 fr...

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Abstract

The invention discloses a zymomonas mobilis-escherichia coli shuttle vector pSUZM1, pSUZM2 and pSUZM3 and a construction method thereof. The zymomonas mobilis-escherichia coli shuttle vector is an annular vector; the pSUZM1 contains a replication origin oriC from a zymomonas mobilis chromosome, an escherichia coli plasmid replication origin from a plasmid pUC18 and a kanamycin screening marker gene; the pSUZM2 contains a replication origin from a zymomonas mobilis endogenous plasmid pZZM401, a DNA (Deoxyribose Nucleic Acid) replication protein gene sequence, the escherichia coli plasmid replication origin from the plasmid pUC18 and the kanamycin screening marker gene; the pSUZM3 contains a replication origin from a zymomonas mobilis endogenous plasmid pZZM402, the DNA replication protein gene sequence, the escherichia coli plasmid replication origin from the plasmid pUC18 and the kanamycin screening marker gene. The zymomonas mobilis-escherichia coli shuttle vector disclosed by the invention can not only be replicated in escherichia coli, but also be replicated in zymomonas mobilis, can be stably inherited in the zymomonas mobilis and has very good application prospect.

Description

technical field [0001] The invention relates to Zymomonas mobilis-Escherichia coli shuttle vectors pSUZM1, pSUZM2, pSUZM3 and their construction methods and applications, belonging to the technical field of genetic engineering. Background technique [0002] Energy security and environmental issues have become the focus of the world's common concern, and fuel ethanol, as an excellent alternative energy source, has attracted extensive attention of researchers. Zymomonas mobilis (Zymomonasmobilis, Z.mobilis) has attracted more and more attention in the field of biotechnology. It can not only be used to produce important chemical products, but also can be used to produce fuel ethanol. Compared with Saccharomyces cerevisiae and other fuel ethanol genetically engineered bacteria, Zymomonas mobilis has many outstanding advantages, such as high sugar absorption efficiency and low production volume; no need to control oxygen addition during fermentation; high osmotic pressure resista...

Claims

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Application Information

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IPC IPC(8): C12N15/74C12N15/66
Inventor 谭雪梅曹庆华张义正王海燕冯红
Owner SICHUAN UNIV
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