Part:BBa_K5072002
P57-1
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_K5072002 (P57-1)
Name: P57-1
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
We designed the 20-base sequence on the primer, and used the complete pEcgRNA-P27-1 as a template for reverse PCR amplification to replace the N20 sequence to obtain a complete new recombinant plasmid. The 20-base sequence was synthesized by a biological company.
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