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A genetic manipulation strategy of Hansenula polymorpha and its application

A Hansenula polymorpha, a part of the technology, applied in the field of Hansenula polymorpha genetic manipulation strategy, can solve the problems of cell lethal effect, high false positive rate, low screening efficiency, etc., to achieve a wide range of applications and overcome low efficiency. Effect

Active Publication Date: 2016-05-04
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this reverse screening method is only limited to ura3-deficient or trp1-deficient Hansenula cells, and the false positive rate is high, and the screening efficiency is low
[0006] The product encoded by the mazF gene of Escherichia coli can specifically recognize and cut the 5'-ACA-3' sequence of mRNA, thereby producing a lethal effect on cells. As a toxic protein, whether it can be used as a reaction in the genetic manipulation of Hansenula polymorpha Screening markers have not been reported yet

Method used

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  • A genetic manipulation strategy of Hansenula polymorpha and its application
  • A genetic manipulation strategy of Hansenula polymorpha and its application
  • A genetic manipulation strategy of Hansenula polymorpha and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1. Expression and biological function analysis of Escherichia coli toxic protein mazF

[0063] 1. Construction of recombinant plasmid pMOXZ-mazF

[0064] (1) According to the mazF sequence of Escherichia coli JM109 toxic protein gene (GenBankAccessNo.U00096.2), the following primers were designed and synthesized:

[0065] Primer 1: 5'-CGG AAGCTT ATGGTAAGCCGATACGTAC-3';

[0066] (The underlined part is the recognition site of HindⅢ enzyme digestion)

[0067] Primer 2: 5'-CCC TCTAGA AGTAACACTACCCCAATCAGT-3'.

[0068] (The underlined part is the recognition site of XbaⅠ digestion)

[0069] (2) Extract the genomic DNA of Escherichia coli JM109, use the genomic DNA as a template, and perform PCR amplification with primers 1 and 2.

[0070] PCR reaction system: Genomic DNA 50ng, primer 1 final concentration 0.3μmol / L, primer 2 final concentration 0.3μmol / L, KOD-Plus-NeoDNA polymerase 1μL, 10×KODbuffer 5μL, 2mMdNTPs 5μL, 25mMMg 2+ 2 μL, make up the system to 5...

Embodiment 2

[0093] Example 2, application of mazF expression cassette and zeoR expression cassette in gene marker-free knockout

[0094] 1. Construction of recombinant plasmid pEBCMMZC

[0095] (1) According to the CYC1TT sequence on the vector pMOXZα-A, design the following primers:

[0096] Primer 3: 5'-ATCCAATTGTGACACGTCCGAC-3';

[0097] Primer 4: 5'-CCTTTTACGGTTCCTGGCC-3'.

[0098] (2) Using pMOXZα-A as a template, PCR was performed with primers 3 and 4 to amplify the CYC1TT fragment.

[0099] PCR reaction system: pMOXZα-A50ng, primer 3 final concentration 0.3μmol / L, primer 4 final concentration 0.3μmol / L, KOD-Plus-NeoDNA polymerase 1μL, 10×KODbuffer 5μL, 2mMdNTPs 5μL, 25mMMg 2+ 2 μL, make up the system to 50 μL with deionized water, and mix well.

[0100] PCR reaction conditions: 94°C for 5 minutes, 1 cycle; 94°C for 30 seconds, 56°C for 30 seconds, 68°C for 20 seconds, 30 cycles; 68°C for 10 minutes, 1 cycle.

[0101] (3) After the PCR product was detected by agarose gel electr...

Embodiment 3

[0192] Example 3, application of mazF expression cassette and zeoR expression cassette in gene traceless knockout

[0193] 1. Amplification of PEP45'UP and mazF-zeoR

[0194] (1) According to the PEP4 gene sequence and the mazF-zeoR sequence in pMOXZ-mazF, the following primers were designed and synthesized:

[0195] Primer 15: 5'-AAA GAGCTC TCGACGCGGAGAACGATCTC-3';

[0196] (The underlined part is the recognition site of SacⅠ enzyme digestion)

[0197] Primer 16: 5'-GATGAGCATTCAGAGCTGTTGCAAATTAAAGCCTTCGAGC-3';

[0198] Primer 17: 5'-GCTCGAAGGCTTTAATTTGCAACAGCTCTGAATGCTCATC-3';

[0199] Primer 18: 5'-AAA GGTCACC AGCTCGGCCCGCCAGAAAT-3'.

[0200] (The underlined part is the recognition site of BstEⅡ restriction enzyme)

[0201] (2) Using pMOXZ-mazF as a template, PCR amplification was performed using primers 15 and 16 to obtain a 3.5kb sequence of mazF-zeoR, which is the first to the 3449th core from the 5' end of SEQIDNo.6 nucleotides are shown.

[0202] PCR reaction...

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Abstract

The invention discloses a hansenula polymorpha genetic operation strategy and application thereof. The hansenula polymorpha genetic operation strategy is characterized in that a DNA (Deoxyribonucleic Acid) fragment group used for knocking out a target gene of hansenula polymorpha consists of a DNA fragment A and a DNA fragment B; the DNA fragment A in the direction from end 5' to end 3' is structured as 5' end sequence of the target gene, m, mazF expression cassette, and part of resistance screening marker gene cassette fragment A, in sequence; the DNA fragment B in the direction from 5' end to 3' end is structured as part of resistance screening marker gene cassette fragment B, n, and 3' end sequence of the target gene, in sequence. According to the hansenula polymorpha genetic operation strategy, methanol induced toxic protein mazF has the effect of cutting mRNA (messenger Ribonucleic Acid), thus the purpose of knockout without a marker or a trace can be realized; the hansenula polymorpha genetic operation can be performed conveniently.

Description

technical field [0001] The invention relates to a genetic manipulation strategy of Hansenula polymorpha and its application. Background technique [0002] Hansenula polymorpha (Hansenula polymorpha) is a kind of methanol-trophic yeast that can grow rapidly with methanol as the only carbon source and energy source. It has gene expression regulation and protein translation similar to higher eukaryotes like other yeasts In addition to the post-modification mechanism, it also has a unique mechanism that is beneficial to the high-efficiency expression of foreign proteins. For example, the endogenous methanol oxidase (methanoloxidase) gene promoter MOXp is a strong inducible promoter, which is not sensitive to the repression of glucose or glycerol. Under the condition of limitation or lack of glycerol, it can be induced by methanol to start the high-efficiency expression of exogenous genes, so cell culture and protein expression can be controlled by time, and the controllability o...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12N15/81C12N1/19C12R1/78
Inventor 何秀萍宋盼盼郭雪娜刘莎张博润
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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