Team:East Chapel Hill/Composite Part

Best New Composite Part

We have one new composite part: the Fluoride Riboswitch Regulated Chloramphenicol Acetyltransferase Operon.

Schematic of the fluoride riboswitch regulated chloramphenicol acetyltransferase operon (CHOP).

Promoter used: BBa_J23100.
The restriction enzymes sites make it easy to put in new genes using the Gibson Assembly just order your gene of interest with the correct gibson homology overhangs (15-20nts) and cut with the appropriate restriction enzyme.

HindIII = promoter riboswitch segment
XhoI = new genes

This part allows for determining the responsiveness of a fluoride riboswitch and can be used to qualitatively measure fluoride levels. How it works:

  • Using the ΔcrcB E. coli strain, which can accumulate fluoride intracellularly.
  • The Riboswitch detects fluoride Fluoride activates the chloramphenicol acetyltransferase enzyme, which allows for the growth of bacteria on agar plates with the antibiotic chloramphenicol.

This system is amenable for high-throughput screening and can be used to identify fluoride riboswitches with higher affinity to fluoride. However, this system should work with any transcriptional riboswitch.