FLP/FRT gene knockout method of Candida amazonensis

A gene knockout and gene technology, applied in the field of genetic engineering, can solve problems such as differences in the function of promoters, and achieve the effect of eliminating the screening process, eliminating hygromycin resistance and normal morphology

Active Publication Date: 2017-05-10
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, promoters from different sources or different expression h...

Method used

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  • FLP/FRT gene knockout method of Candida amazonensis
  • FLP/FRT gene knockout method of Candida amazonensis
  • FLP/FRT gene knockout method of Candida amazonensis

Examples

Experimental program
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Embodiment 1

[0057] Example 1 Site-directed mutation of the recombinase gene FLP

[0058] Using Saccharomyces cerevisiae as a template, PCR amplification with primers FLP-F (SEQ ID NO: 19) and FLP-R (SEQ ID NO: 20) was used to obtain the FLP gene sequence (enzyme cleavage site Sal I was introduced upstream of the sequence, and enzyme cleavage site was introduced downstream of the sequence cut site Not I), connected to the PMD 19-T simple vector, transformed into competent E.coli JM109, and obtained the recombinant Ts-FLP. Using the plasmid Ts-FLP as a template, the Stratagene site-directed mutagenesis kit (purchased from Agilent Technologies Co., Ltd.) was used, and primers FLP-1 (SEQ ID NO: 21) and FLP-2 (SEQ ID NO: 22); FLP- 3 (SEQ ID NO: 23) and FLP-4 (SEQ ID NO: 24); FLP-5 (SEQ ID NO: 25) and FLP-6 (SEQ ID NO: 26) undergo three rounds of PCR site-directed mutagenesis to obtain circular like PCR products. The amplification conditions were: 95°C pre-denaturation for 2 minutes, 95°C den...

Embodiment 2

[0059] The construction of embodiment 2PDC gene knockout cassette

[0060] (1) Using the Candida amazonensis genome as a template, use primers PDC-1 (SEQ ID NO: 8) and PDC-2 (SEQ ID NO: 9) to amplify the upstream sequence PDC-L of the 5' end of the PDC gene 360bp, in the fragment The restriction site Stu I was introduced upstream, and the restriction site Kpn I was introduced downstream of the fragment; primers PDC-3 (SEQ ID NO: 10) and PDC-4 (SEQ ID NO: 11) were used to amplify 400 bp of the PDC 3' end The downstream sequence of PDC-R, the restriction site Kpn I was introduced upstream of the fragment, and the restriction site Stu I was introduced downstream of the fragment; the PDC-L and PDC-R sequences were spliced ​​by overlap extension PCR to obtain the PDC gene homologous recombination fragment PDCL -R.

[0061] (2) Connect PDCL-R to PMD19-T simple vector, transform competent E.coli JM109, obtain a positive clone, and name it Ts-PDCL-R. The plasmid Ts-PDCLR was extract...

Embodiment 3

[0067] Embodiment 3 PEG / LiAc method transforms Candida amazonensis CBS 12363

[0068](1) Pick a single colony of Candida amazonensis CBS 12363 from a YPD plate, inoculate it in 20 mL of YPD medium, and culture it overnight at 30°C in a 100 mL shake flask;

[0069] (2) Inoculate the overnight cultured fresh bacterial solution into 50mL YPD medium, and culture it in a 250mL shake flask at 30°C and 200rpm until the bacterial solution OD 600 to around 1.2;

[0070] (3) Centrifuge at room temperature for 5 minutes at 5000 rpm to collect bacterial cells;

[0071] (4) Resuspend the cells in 500 μL of 0.1mol / L LiAc, centrifuge, discard the supernatant, and obtain competent cells;

[0072] (5) Add the following transformation mixture in order to the competent cells: 240 μL 50% PEG3350, 36 μL 1.0mol / L LiAc, 25 μL salmon sperm DNA, 50 μL linear DNA to be transformed, among which the salmon sperm DNA is in boiling water for 10 minutes and then ice-bathed immediately ;

[0073] (6) Sha...

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Abstract

The invention discloses an FLP/FRT gene knockout method of Candida amazonensis and belongs to the technical field of gene engineering. The FLP/FRT gene knockout method comprises the following steps: putting an FLP recombinase gene under a strictly induced type promoter SpGAL1p or SpMAL1p and regulating and controlling; adding directly repeated FPT sites to two ends of an FLP expression box and a resistance marker expression box; constructing a gene knockout box by taking PDC as a target gene and integrating the gene knockout box into a Candida amazonensis genome; then, adding an induction agent to induce FLP expression, so as to realize excision of resistance genes. According to the FLP/FRT gene knockout method disclosed by the invention, marker-free gene knockout of the Candida amazonensis is realized for the first time, and the method can be used for continuously knocking out a plurality of genes; a powerful technical means is provided for modification of metabolism engineering of the Candida amazonensis.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a Candida amazonensis FLP / FRT gene knockout system. Background technique [0002] Candida amazonensis is a yeast that can efficiently utilize xylose isolated from the Amazon forest in recent years. Lopes MR, Pereira GM, Zilli JE, Vital MJ, Gomes FC, Lachance MA, Rosa CA. Candida amazonensis sp.nov., an ascomycetous yeast isolated from rottingwood in the Amazonian forest. International journal of [0003] systematic and evolutionary microbiology, 2012, 62(Pt 6): 1438-1440.). Cadete et al. (Cadete RM, Melo MA, Dussan KJ, Rodrigues RC, Silva SS, Zilli JE, Vital MJ, [0004] Gomes FC, Lachance MA, Rosa CA. Diversity and physiological characterization of D-xylose-fermenting yeasts isolated from the Brazilian Amazonian Forest. PLoS [0005] ONE,2012,7(8):e43135.) found that Candida amazonensis has the ability to metabolize xylose rapidly and has certain advantages in xylitol produ...

Claims

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Application Information

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IPC IPC(8): C12N15/81C12N15/66C12R1/72
CPCC12N9/88C12N15/66C12N15/815C12N2800/30C12Y401/01001
Inventor 张梁高芝顾正华李由然丁重阳石贵阳
Owner JIANGNAN UNIV
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