Team:Calgary/FLP-Beta
FLP-BETA
FLP Recombinase & Beta Resolvase Use Overview
What is FLP Recombinase?
In order to introduce the plasmid construct (BBa_K2605006) into the genome, utilising the CRISPR knock-in landing pad, FLP recombinase (FLP) was used. FLP binds to specific sites named FRT (flippase recombination target) sites. When FLP recognizes two target sites that have matching sequences, FLP binds, cleaving both sites allowing for a “crossing over” event to take place in which FLP ligates the two sequences together. While the exact mechanism is hard to visualise at the nucleotide level, it is simply understood by comparing the before and after of the recombination event.
In our system an FRT site is found within the genome of our modified HEK293T cells and another, complimentary, FRT site is located on the plasmid construct (BBa_K2605006), along with another plasmid (pCAG-FLPo) containing the gene coding for FLP. Once the plasmids are introduced to the HEK293T cells pCAG-FLPo transiently expresses FLP, which then recognizes the complimentary FRT sites. When FLP cleaves and ligates these sites, the entire plasmid construct is integrated into the genome, thus the genome now contains all genetic elements from both the CRISPR knock-in and the plasmid construct.
This recombination is supplemented by the 𝛽-resolvase (secondary recombination event), which removes the possibility of FLP naturally excising the inserted plasmid construct through the reverse reaction, which is favorable.