Part:BBa_K5072003
P57-2
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
BBa_K5072003 (P57-2)
Name: P57-2
Base Pairs: 20 bp
Origin: https://benchling.com/
Usage and Biology
The 20-base gRNA sequence designed by Benchling is derived from the precise design of the DNA sequence of the T7 phage P57 gene. The purpose is to bind to the constitutive Cas9 protein to cleave the T7 phage P57 gene. The arrangement of these bases determines the specificity and cleavage efficiency of gRNA.
Its key attributes include base composition, GC content, sequence specificity, potential off-target effect, secondary structure stability, and PAM site dependence (such as NGG), which together affect the activity and reliability of gRNA. Reasonable gRNA design can minimize off-target effects and improve editing efficiency, which is a key factor for the success of the CRISPR-Cas9 system.
We designed a 20-base gRNA sequence to bind to the constitutive Cas9 protein and target and cleave the P57 gene of T7 phage, which plays an important role in phage replication and infection. By targeting the P57 gene, the CRISPR-Cas9 system can effectively prevent the replication of T7 phage and enhance the anti-phage ability of host bacteria (such as E. coli MG1655).
Such strategies are widely used in the development of engineering bacteria against phage infection, which helps to enhance the stability of bacteria in industrial fermentation and drug production.
Cultivation
By directly synthesizing the target 20-base sequence and using the pEcgRNA-P29-2 plasmid preserved in the laboratory as a template, we designed primers to replace its N20 part. The recombinant plasmid containing the target sequence was obtained by reverse PCR amplification of the plasmid fragment bound by the primer.
This method realized the rapid construction of gRNA sequences through precise primer design and efficient PCR amplification, which provided a basis for the subsequent application of the CRISPR-Cas9 system.
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