Team:British Columbia/Parts

BBa_K2700000

The FLS1 gene encodes a catalytic protein that aids in the biosynthetic pathway to form kaempferol. The protein can both form dihydrokaempferol from naringenin through catalyzing oxidation and use dihydrokaempferol to produce kaempferol. The FLS1 protein is also a ligand that binds to iron.

Biosensor Parts

Introduction

The pT181 transcript-dependant transcriptional and translational regulation mechanism was adapted from the Staphylococcus aureus plasmid pT181 [1]. In Staphylococcus aureus this system regulates the expression of RepC, which is responsible for the replication of the pT181 plasmid [1]. The regulation system contains a promoter and the associated 5’ untranslated region (BBa_K2700002), as well as a cis-encoded antisense transcript (BBa_K2700001). When expressed, the antisense transcript interacts with the 5’ untranslated region of the pT181-promoter, producing two hairpin structures: a transcriptional terminator and a ribo-switch, blocking the ribosomal binding site [2].

pT181-promoter The pT181-promoter and 5’ untranslated region enable the expression of a downstream gene. In the absence of the antisense transcript, the 5’ region folds to create the structure depicted in figure 1 [2]. In this conformation, both transcription and ribosomal binding are unhindered.

In the presence of the antisense transcript, the 5’ region folds to create the structure depicted in figure 2 [2]. In this conformation, two hairpin structures are formed. The first hairpin structure functions as a premature transcriptional terminator, regulating expression at the transcriptional level. The second hairpin structure is formed at the RBS, regulating expression at a translational level. This dual regulation enabled a 98% repression a RFP expression upon induction of the antisense transcript [2].

BBa_K2700001

This is our pt181-antisense-RNA transcript that will block both transcription and translation at the pt181 promoter (Part:BBa_K2700002) site. Once pt181-antisense is expressed, it will bind to the promoter region and cause a physical fold to occur. This fold is a stem-like structure that will block the RBS as well as act as a terminator to prevent further transcription to occur.

BBa_K2700002

This is our pt181-promoter region for our biosensor.