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Construction and application of Zymomonas mobilis CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-association proteins)9 system

A Zymomonas, psuzm1a-cas9 technology, applied in the construction and application of Zymomonas mobilis CRISPR-Cas9 system, can solve the problems of complex experimental process and low efficiency, and achieve the effect of good application prospects

Inactive Publication Date: 2014-10-22
SICHUAN UNIV
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  • Description
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AI Technical Summary

Problems solved by technology

Conventional plasmid elimination methods include physical and chemical methods, but the efficiency of these methods is not high, and the experimental process is relatively complicated

Method used

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  • Construction and application of Zymomonas mobilis CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-association proteins)9 system
  • Construction and application of Zymomonas mobilis CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-association proteins)9 system
  • Construction and application of Zymomonas mobilis CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-association proteins)9 system

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0045] Example 1 Construction of expression plasmid pUC-T7sgRNA

[0046] 1) Design of T7sgRNA gene: Artificially synthesize a double-stranded DNA with a full length of 177bp, including T7 gene promoter and terminator, Bbs I recognition sequence and crRNA-tracrRNA sequence, the two ends of the sequence are respectively Hin dIII and Eco RI site.

[0047] The T7sgRNA gene sequence is as follows:

[0048] GG AAGCTT AA TACGACTCAC TATAGGTCTT CGA GAAGAC C TGTTTTAGAG CTAGAAATAG CAAGTTAAAA TAAGGCTAGT CCGTTATCAA CTTGAAAAAG TGGCACCGAG TCGGTGCTTT TTCTAGCATA ACCCCTTGGG GCCCTTAAAC GGGTCTTGAG GGGTTTTTT G AATTC CC

[0049] 2) Plasmid pUC19 and T7sgRNA gene fragments were used respectively Hin dIII and Eco RI for double digestion

[0050] 3) The digested product was ligated with T4 DNA ligase, transformed into Escherichia coli, and the expression plasmid pUC-T7sgRNA was obtained (see figure 1 ).

example 2

[0051] Construction of example 2 expression plasmids pSUZM1a-Cas9, pSUZM2a-Cas9, pSUZM3a-Cas9

[0052] For the construction strategy of expression plasmids pSUZM1a-Cas9, pSUZM2a-Cas9, pSUZM3a-Cas9, see figure 2 , image 3 , Figure 4 .

[0053] 1) Design primers 1V-Cas9 upstream, 1V-Cas9 downstream; primer Cas9-1 upstream, primer Cas9-1 downstream; the specific sequence is as follows:

[0054] 1V-Cas9 upstream: 5'-CTAGGAGGTGACTGAAGGTAGCTTGCAGTGGG-3'

[0055] 1V-Cas9 downstream: 5'-GAGTATTTCTATCCATTGCTTACTCCATATAT-3'

[0056] Cas9-1 upstream: 5'-ATATATGGAGTAAGCA ATGGATAAGAAATACTC-3'

[0057] Downstream of Cas9-1: 5'-CCCACTGCAAGCTACCT TCAGTCACCTCCTAG-3'

[0058] 2) Using plasmids pSUZM1a, pSUZM2a, and pSUZM3a as templates, use primers 1V-Cas9 upstream and 1V-Cas9 downstream to amplify vector backbone fragments A, B, and C; use Streptococcus pyogenes CICC10464 as a template; use primer Cas9-1 upstream and downstream of Cas9-1 to amplify the Cas9 gene fragment.

[0059] P...

example 3

[0076] Example 3 Application of CRISPR-Cas9 system in Escherichia coli

[0077] 1) Transform Escherichia coli DH5α with plasmids pSUZM1a-Cas9, pSUZM2a-Cas9, pSUZM3a-Cas9

[0078] Preparation of competent cells: Pick a single colony of Escherichia coli strain DH5α that was cultured overnight, inoculate it in 2 mL of SOB, and inoculate it with shaking at 37 °C overnight; take 0.5 mL of the bacterial liquid, inoculate it in 50 mL of SOB, and inoculate it at 18 °C for about 24 h or shake culture at 25°C for about 12 h; transfer the bacterial solution into a 50 mL centrifuge tube, bathe in ice water for 10 min, centrifuge at 4,000 rpm for 10 min, discard the supernatant; add 16 mL of 0°C pre-cooled TB buffer ( 20 mmol / L KCl, 54 mmol / L MnCl 2 , 15 mmol / L CaCl 2 , 12.5 mmol / L K-MES pH 6.2), resuspend the cells, centrifuge at 4,000 rpm for 10 min, then add 4 mL of TB buffer to suspend the cells, add 280 μL DMSO dropwise, mix well, and place in an ice bath for 10 minutes min or long...

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Abstract

The invention discloses construction of CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-association proteins) system expression plasmids pSUZM1a-Cas9, pSUZM2a-Cas9 and pSUZM3a-Cas9. The CRISPR-Cas system expression plasmids comprise a Zymomonas mobilis endogenous gene promoter, a selective marker gene and a Cas9 gene of a CRISPR system. The construction method comprises the following steps: constructing an expression plasmid pUC-T7sgRNA (single-guide ribonucleic acid) comprising an origin of replication, the selective marker gene, a T7 gene promoter and terminator, a BbsI recognition sequence and a crRNA-tracrRNA sequence; designing a corresponding target sequence to ensure that a DNA template is subjected to in vitro transcription by virtue of T7RNA polymerase, and sgRNA and Cas9 gene expression plasmids obtained by purification are used for performing co-electrotransformation on escherichia coli and Z.mobilisZM4. Results show that an upp gene of escherichia coli DH5alpha can be successfully knocked out by adopting a CRISPR technology, and natural plasmids in Zymomonas mobilis can be effectively removed. The Cas9 gene expression plasmid, the sgRNA expression plasmid and a complete set of research methods established in the research process can be widely applied to the knockout of genes in a Zymomonas mobilis genome, thus the construction of a Zymomonas mobilis CRISPR-Cas9 system disclosed by the invention has good market application prospects.

Description

technical field [0001] The invention relates to the construction of Zymomonas mobilis CRISPR-Cas9 expression plasmids pSUZM1a-Cas9, pSUZM2a-Cas9, pSUZM3a-Cas9, pUC-T7sgRNA and the application of CRISPR-Cas9 system in Escherichia coli and Zymomonas mobilis, belonging to gene field of engineering technology. Background technique [0002] Zymomonas mobilis has high ethanol yield and high sugar absorption efficiency; strong acid and ethanol tolerance; no need to control oxygen addition during fermentation; high osmotic pressure resistance; small genome (about 2Mbp), easy to carry out gene (group) engineering (gene or genome engineering) breeding and many other advantages. However, at present, Zymomonas mobilis has not been widely used in ethanol fermentation production on a large scale. The main limiting factors are: it cannot convert complex carbohydrate polymers such as cellulose, hemicellulose and starch into ethanol; A variety of by-products, such as sorbitol, 3-hydroxybut...

Claims

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Application Information

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IPC IPC(8): C12N15/74C12N15/66C12R1/01
Inventor 谭雪梅曹庆华张义正王海燕冯红
Owner SICHUAN UNIV
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