Series of Zymomonas mobilis expression vectors pSUZM, and construction method thereof

A technology of Zymomonas and expression vectors, which is applied in the field of expression vectors and can solve the problems of lack of genetic engineering strains and limited applications

Inactive Publication Date: 2014-12-17
SICHUAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] The deficiency of Zymomonas mobilis limits its application in the fields of basic resea...

Method used

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  • Series of Zymomonas mobilis expression vectors pSUZM, and construction method thereof
  • Series of Zymomonas mobilis expression vectors pSUZM, and construction method thereof
  • Series of Zymomonas mobilis expression vectors pSUZM, and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0072] Example 1 Construction of expression vectors pSUZM1a, pSUZM1b, pSUZM1c

[0073] For the construction strategy of expression vector pSUZM1a, pSUZM1b, pSUZM1c, see figure 1 .

[0074] 1) Design the upstream primer P1_pdc F, downstream primer P1_pdc R of the vector backbone A; the upstream primer 1_pdc F, downstream primer 1_pdc R of the Pdc promoter; the upstream primer P1_ara F of the vector backbone B, the downstream primer P1_ara R; the upstream of the Pdc promoter Primer 1_ara F, downstream primer 1_ara R, upstream primer P1_tac F, downstream primer P1_tac R of vector backbone C; upstream primer 1_tac F, downstream primer 1_tac R of the Pdc promoter, for specific primer sequences, see figure 2 .

[0075] 2) Using the vector pSUZM1 as a template, use P1_pdcF / P1_pdc R, P1_ara F / P1_ara R, P1_lacF / P1_lac R as primers to amplify the vector backbone fragments A, B and C respectively; using the whole genome DNA of Zymomonas mobilis as a template, Use 1_pdc F, 1_pd...

example 2

[0089] Example 2 Construction of expression vectors pSUZM2a, pSUZM2b, pSUZM2c

[0090] For the construction strategy of expression vector pSUZM2a, pSUZM2b, pSUZM2c, see Figure 4 .

[0091] 1) Design the upstream primer P2_pdc F and downstream primer P2_pdc R of the vector backbone D; the upstream primer 2_pdc F and downstream primer 2_pdc R of the Pdc promoter; the upstream primer P2_ara F and downstream primer P2_ara R of the vector backbone E; the upstream primer of the Pdc promoter 2_ara F, downstream primer 2_ara R, upstream primer P2_tac F of the vector backbone F, downstream primer P2_tac R; upstream primer 2_tac F of the Pdc promoter, downstream primer 2_tac R, for specific primer sequences see Figure 5 .

[0092] The vector pSUZM2 was used as a template, and P2_pdcF / P2_pdc R, P2_ara F / P2_ara R, P2_tacF / P2_tac R were used as primers to amplify vector backbone D, E and F fragments, respectively. Using the whole genome DNA of Zymomonas mobilis as template, Ppdc-2 was...

example 3

[0105] Example 3 Construction of expression vectors pSUZM3a, pSUZM3b, pSUZM3c

[0106] For the construction strategy of expression vector pSUZM2a, pSUZM2b, pSUZM2c, see Figure 6 .

[0107] 1) Design the upstream primer P3_pdc F and downstream primer P3_pdc R of vector backbone G; the upstream primer 3_pdc F and downstream primer 3_pdc R of the Pdc promoter; the upstream primer P3_ara F and downstream primer P3_ara R of the vector backbone H; the upstream primer of Pdc promoter 3_ara F, downstream primer 3_ara R, upstream primer P3_tac F, downstream primer P3_tac R of the vector backbone I; upstream primer 3_tac F, downstream primer 3_tac R of the Pdc promoter, for specific primer sequences see Figure 7 .

[0108] 2) Using the vector pSUZM3 as a template, use P3_pdcF / P3_pdc R, P3_ara F / P3_ara R, P3_lacF / P3_lac R as primers to amplify vector backbone G, H and I fragments respectively. Using the whole genome DNA of Zymomonas mobilis as template, Ppdc-3 was amplified with 3_p...

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Abstract

The invention discloses a series of expression vectors pSUZM1a, pSUZM1b, pSUZM1c, pSUZM2a, pSUZM2b, pSUZM2c, pSUZM3a, pSUZM3b and pSUZM3c, and a construction method thereof. The expression vectors are annular vectors, and respectively contain replication origin from Escherichia coli plasmid on plasmid pUC18, and a kanamycin screening marking gene, wherein pSUZM1(a, b, c) respectively contain replication origin oriC from Zymomonas mobilis chromosome, pSUZM2(a, b, c) respectively contain replication origin from Zymomonas mobilis endogenous plasmid pZZM401, and the pSUZM3(a, b, c) respectively contain replication origin from Zymomonas mobilis endogenous plasmid pZZM402. The pSUZM1a, pSUZM2a and pSUZM3a respectively contain promoter Ppdc from Zymomonas mobilis; pSUZM1b, pSUZM2b and pSUZM3b respectively contain promoter Para from vector pKD46; and pSUZM1c, pSUZM2c and pSUZM3c respectively contain tac promoter from vector pGEX. The method realizes the efficient expression of exogenous gene in Zymomonas mobilis, and has a very good application prospect.

Description

technical field [0001] The invention relates to expression vectors pSUZM1a, pSUZM1b, pSUZM1c; pSUZM2a, pSUZM2b, pSUZM2c; pSUZM3a, pSUZM3b, pSUZM3c and their construction methods, belonging to the technical field of genetic engineering. Background technique [0002] Zymomonas mobilis (Zymomonas mobilis) is an ethanol-producing bacterium with great potential for industrial development. Its advantages are high ethanol yield, low biomass production, strong acid and ethanol tolerance, etc. However, it also has Some deficiencies, such as the narrow substrate range, cannot utilize complex carbohydrates for conversion to ethanol. In addition, the bacteria are sensitive to salts, pH, and inhibitors in the mash during fermentation, so the production process needs to be strictly controlled. [0003] The deficiency of Zymomonas mobilis limits its application in the fields of basic research and biotechnology, and one of the main bottlenecks is the lack of genetically engineered strains....

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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