gRNA for targeted reduction of drug-resistant gene blaTEM and drug-resistant plasmid thereof, transferable knockout vector and application of gRNA and transferable knockout vector
A drug-resistant gene, knockout vector technology, applied in DNA/RNA fragments, applications, genetic engineering, etc., can solve the problems affecting the spread of multi-drug resistant plasmids, human and animal health risks, and difficult to kill antibiotics, and reduce The effect of multidrug resistance plasmids
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Embodiment 1
[0047] Example 1 Construction and verification of horizontally transferable gene knockout vector pCas9-Mob
[0048] (1) Using the pCas9 plasmid as a template, design specific primers, use high-fidelity PCR technology to amplify the replicon, screening marker gene and knockout protein gene Cas9 sequence, and obtain OriV-Chl-Cas9 after gel purification.
[0049] (2) Using the plasmid carrying the transfer site as a template, design specific primers, use high-fidelity PCR technology to amplify the transfer site of the plasmid, and obtain OriT after gel purification.
[0050] (3) Using the Novoprotein seamless cloning reagent, connect the above-mentioned purified OriV-Chl-Cas9 and OriT fragments according to the protocol.
[0051] (4) Using CaCl 2 The competent method was prepared, and the above ligation product was transformed into a specific Escherichia coli competent, and the relevant genes required for the horizontal transfer of the pCas9-Mob plasmid were integrated on the ch...
Embodiment 2
[0056] Embodiment 2 constructs targeted drug resistance gene bla TEM and carry bla TEM Transferable Knockout Vectors of Multidrug Resistance Plasmids
[0057] (1) Download drug resistance gene bla from NCBI database TEM Sequence, search for PAM sites, and select gRNAs with GC ratio, specificity and other indicators that meet the requirements from the gRNAs close to the PAM sites, generally 20-25bp is appropriate. The preferred gRNA in this case is:
[0058] gRNA TEM -1: 5'-ATCGAACTGGATCTCAACAG-3'
[0059] gRNA TEM -2: 5'-ACAATTAATAGACTGGATGG-3'
[0060] (2) Design gRNA separately TEM -1 and gRNA TEM The sequence of the annealing primer involved in -2 is as follows:
[0061] gRNA TEM -1 forward primer: 5'-AAACATCGAACTGGATCTCAACAGG-3'
[0062] gRNA TEM -1 reverse primer: 5'-AAAACCTGTTGAGATCCAGTTCGAT-3'
[0063] gRNA TEM -2 forward primer: 5'-AAACACAATTAATAGACTGGATGGG-3'
[0064] gRNA TEM -2 reverse primer: 5'-AAAACCATCCAGTCTATTAATTGT-3'
[0065] (3) Phosphorylat...
Embodiment 3
[0072] Example 3 Verification of gRNA using in vitro targeted cleavage experiments TEM -1 and gRNA TEM -2 pairs of bla TEM the effectiveness of
[0073] (1) Design and synthesize forward primer gRNA TEM -1-F and gRNA TEM -2-F, the sequence is as follows:
[0074] gRNA TEM -1-F:
[0075] 5'-TTAATACGACTCACTATAGGATCGAACTGGATCTCAACAGGTTTTTAGAGCTAGAAATAG-3'
[0076] gRNATEM -2-F:
[0077] 5'-TTAATACGACTCACTATAGGACAATTAATAGACTGGATGGGTTTTTAGAGCTAGAAATAG-3'
[0078] (2) Add 10ul of 2×sgRNA reaction buffer, 2ul of the above forward primer, 2ul of enzyme mixture, 6ul of sterile water into the 20ul system, transcribe in vitro for 1h to obtain sgRNA TEM -1 and sgRNA TEM -2.
[0079] (3) Take an appropriate amount of purified bla TEM Gene fragment, add 2ul reaction buffer, 1ul Cas9 enzyme mixture, 1ul above sgRNA TEM , and make up to 20ul with sterile water, react at 37°C for 1h, and react at 70°C for 10min.
[0080] (4) The above bla TEM Gene fragment, via sgRNA TEM -1 and...
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