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
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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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