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.
The pT181 transcript-dependant transcriptional and translational regulation mechanism was adapted from the Staphylococcus aureus plasmid pT181 . In Staphylococcus aureus this system regulates the expression of RepC, which is responsible for the replication of the pT181 plasmid . 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 .
pT181-promoter The pT181-promoter and 5’ untranslated region enable the expression of a downstream gene. In the absence of the antisense transcript, both transcription and ribosomal binding are unhindered.
In the presence of the antisense transcript, the 5’ region folds to create two hairpin structures. 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 .
pT181 Testing protocol
In order to test the repression of GP2 expression by the pT181-antisense transcript, when GP2 is under the control of the pT181-promoter, cells were cultured at various pT181-antisense induction levels. Uninduced cells are expected to grow more slowly, as a result of uncontrolled GP2 expression. Increasing induction level should lead to an increase in growth rate, which will be analyzed in a plate reader by measuring OD600.
1. Co-transform IPTG-inducible pT181-antisense transcript, in a pET15b vector, and pT181-GP2 construct, in a pSB1C3 vector, into BL21 chemically competent cells.
2. Plate on chloramphenicol (Cm) and and ampicillin (Amp) to select for colonies that have taken up both plasmids.
3. In 96-well plate, inoculate cultures in 200ul of LB + Cm + Amp.
4. Shake overnight at 37C.
5. In 96-well plate, transfer 5ul of overnight culture in 200ul of LB + Cm + Amp at 0mM, 0.05mM and 0.5mM of IPTG.
6. Measure OD600 at 1 hour time intervals for 8 hours.