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Method for constructing non-resistance mark auxotroph torulopsis glabrata

A technology of Togus glabrata and auxotrophs, which is applied in the field of bioengineering to achieve the effect of avoiding homologous recombination

Inactive Publication Date: 2011-10-05
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, there are few institutions in China to study the molecular biology of Toructus glabrata, and it is still in the preliminary stage of research

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] According to the method described above, using primers P1 / P2 and P3 / P4, using genomic DNA as a template, and using pfu high-fidelity DNA polymerase, the left and right arms of the ARG8 gene were obtained respectively. The PCR conditions were: 95 °C, 3 min; { 95°C, 50s; 55°C, 50s; 72°C, 1min}, cycle 30 times; 72°C, 15min; store at -20°C.

[0040] The obtained left and right arms were respectively recovered by gel tapping, and then used as templates respectively, and P1 / P4 were used as primers to obtain Δarg8 fragments with large fragment deletions. PCR conditions are as follows: 94°C, 4 min; {94°C, 50s; 55°C, 50s; 72°C, 3min}, cycle 30 times; 72°C, 12min, -20°C for later use.

[0041] The obtained Δarg8 fragment was electrotransformed into T. glabrata CCTCC NO: M 202019, and cultured according to the subsequent enrichment steps to obtain an arginine-deficient T. glabrata strain. The colony PCR was further verified with primers P1 / P4.

[0042] Primer number

Embodiment 2

[0044] According to the method described above, using primers P5 / P6 and P7 / P8, using genomic DNA as a template, and using pfu high-fidelity DNA polymerase, the left and right arms of the URA3 gene were obtained respectively. The PCR conditions are: 95 ° C, 3 min; { 95°C, 50s; 55°C, 50s; 72°C, 2min}, cycle 30 times; 72°C, 15min; store at -20°C.

[0045] The obtained left and right arms were respectively recovered by tapping and then used as templates to obtain a Δura3 fragment with a large fragment deletion. PCR conditions are as follows: 94°C, 4 min; {94°C, 50s; 55°C, 50s; 72°C, 3min}, cycle 30 times; 72°C, 12min; -20°C storage.

[0046] The obtained Δura3 fragment was electrotransformed into T. glabrata CCTCC NO: M 202019, and cultured according to the subsequent enrichment steps to obtain a uracil-deficient T. glabrata strain. The colony PCR was further verified with primers P5 / P8.

[0047] Primer number

Embodiment 3

[0049] The Δura3 fragment obtained in Example 2 was electro-transformed into the arginine-deficient strain obtained in Example 1, and cultured according to the subsequent enrichment steps to obtain an arginine / uracil double-deficient T. glabrata strain. The PCR was further verified with primers P1 / P4 and P5 / P8, respectively.

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Abstract

A constructing method for non-resistance badge auxotrophy smoothing ball false yeast belongs to biological engineering technology field. The invention is a system method for breeding large fragment deletion non-resistance badge auxotrophy smoothing ball false yeast comprising obtaining large fragment deletion consanguinity recombination fragments by blending PCR method, preparing high efficient sprout fungi electric conversion competence, concentrating candio-hermal and screening restrictive culture medium. Goal auxotrophy bacterial strain can be obtained quickly within 3 to 5 days by a suit system method. The deletion of large fragments avoids efficiently reversion and consanguinity recombination of exogenous plasmid. Furthermore, the method can be used many times in a same leaving strain so that multiplex auxotrophy for following genetic engineering study can be obtained because any resistance badge is not used in entire process.

Description

technical field [0001] A method for constructing an auxotrophic T. glabrata without resistance marker, the present invention utilizes fusion PCR, high-efficiency yeast electrotransformation competent preparation, nystatin enrichment and restriction medium screening to construct a large fragment deletion without resistance The invention discloses a sex-marking auxotrophic Toluobacillus glabrata, belonging to the technical field of bioengineering. Background technique [0002] T. glabrata is a haploid yeast with no sexual reproduction stage. T. glabrata is widely used for the production of pyruvate and α-ketoglutarate. Certain strains of T. glabrata are also clinically opportunistic pathogens. The genetic engineering research of T. glabrata is still in its infancy. The most commonly used genetic markers in yeast are auxotrophic markers that enable complementation. [0003] At present, there are few institutions studying the molecular biology of T. glabrata in China, and it...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/19C12R1/645
Inventor 刘立明周景文陈坚堵国成
Owner JIANGNAN UNIV
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