Part:BBa_K2605006

Hybrid FlpO-beta resolvase

Hybrid FlpO-beta resolvase. This part represents a plug-and-play construct to implement the FLP-Beta strategy in a eukaryotic chassis.

Usage and Biology

The coding sequence for FLPo must be added to the same vector or co-delivered with a second. This construct is intended to be subcloned into the MCS of a mammalian expression vector, downstream of a strong promoter and upstream of a terminator. To maximize the efficiency of the system, the expression vector should include a secondary resistance marker (not Puromycin) and an origin of replication to maintain a plasmid population within the cell for as long as is required for the integration reaction to proceed to completion.

This insert contains: -A gene for beta resolvase -Puromycin resistance for selecting modified cells -FLP recombinase recognition target (FRT) - FRT2 -NPS (Nucleosome Positioning Sequences) allowing for nucleosome binding and beta resolvase action on the Six (1 and 2) sites (beta resolvase recognition sequences). The FRT2 site allows FLPo the whole plasmid to be integrated into the genome, which sets the Six1 and 2 sites in line with each other for beta resolvase to act on. There are two Six2 sites with adjacent NPS sites and one Six1 site, which allows for targeted excision. The vector backbone DNA and the FRT2 site once inside the genome are excised, leaving one copy of Six1, Six2, FRT2, and the puromycin resistance gene inside the genome.

A small number of HEK293T cells trasnfected with all components (CRISPR/Cas9 and sgRNA, FLPo-expressing plasmid, and plasmid with hybrid beta-resolvase: BBa_K2605006) successfully grew in puromycin-containing cell media. Puromycin resistance was placed promoter-less on a plasmid which was designed to enter the genome through Flp recombinase. Thus, growth in puromycin indicates the possibility that not only did some cells successfully receive an FRT insert from CRISPR, but they also had our desired plasmid integrated into their genome using our recombinase system at the FRT site. A deeper explanation of this result can be found on the 2018 iGEM Calgary's [http://2018.igem.org/Team:Calgary/FLP-Beta Flp-Beta page.]

Figure 1. Single HEK cell adhering to plate in puromycin antiobiotic, following transfection with all components.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 1107
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 816
  Illegal BsaI.rc site found at 126