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Method for constructing non-resistance mark auxotroph torulopsis glabrata

A kind of glabrata yeast, auxotrophic technology, applied in the field of bioengineering, to avoid the effect of homologous recombination

Inactive Publication Date: 2008-08-13
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 respectively obtained. The PCR conditions were: 95°C, 3min; 95°C, 50s; 55°C, 50s; 72°C, 1min}, cycle 30 times; 72°C, 15min; store at -20°C.

[0040] After the obtained left and right arms were tapped and recovered, they were used as templates respectively, and P1 / P4 were used as primers to obtain the Δarg8 fragment with large fragment deletion. The PCR conditions are as follows: 94°C, 4min; {94°C, 50s; 55°C, 50s; 72°C, 3min}, cycled 30 times; 72°C, 12min, stored at -20°C for later use.

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

[004...

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, respectively, the left and right arms of the URA3 gene were obtained, and the PCR conditions were: 95°C, 3min; 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 tapped and recovered separately, and then used as templates respectively to obtain the Δura3 fragment with large fragment deletion. The PCR conditions are as follows: 94°C, 4min; {94°C, 50s; 55°C, 50s; 72°C, 3min}, cycle 30 times; 72°C, 12min; store at -20°C.

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

[0047] Primer number

Embodiment 3

[0049] The arginine-deficient strain obtained in Example 1 was electrotransformed with the Δura3 fragment obtained in Example 2, and cultured according to the subsequent enrichment steps to obtain an arginine / uracil double-deficient Torulopsis glabrata strain. And further PCR verification was carried out 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 auxotrophic toructus glabrata without resistance markers. The invention utilizes fusion PCR, high-efficiency yeast electrotransformation competent preparation, nystatin enrichment and restriction medium screening to construct a non-resistance-free yeast with large fragment deletion. The invention discloses a sex-marked auxotrophic toructus glabrata, which belongs to the technical field of bioengineering. Background technique [0002] Torulopsis glabrata is a haploid yeast without a sexual reproductive stage. Torulopsis glabrata is widely used for the production of pyruvate and α-ketoglutarate. Certain strains of Torulopsis glabrata are clinically opportunistic pathogens. The genetic engineering research of Torulopsis glabrata is still in its infancy. The most commonly used genetic markers in yeast are auxotrophic markers that can accomplish complementation. [0003] At present, there are few institutions in China to study ...

Claims

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Application Information

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Patent Type & Authority Applications(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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