Main Experimental Results

1. Promoter characterisation

(a) Characterising the CUP1 promoter induction characteristics

Here, we successfully characterised the induction characteristics of the CUP1 promoter using construct CUP1-[GFP]
Timed Induction of the CUP1 Promoter Using CUP1p-GFP
Copper Dose Response of the CUP1 Promoter Using CUP1p-GFP

b) Characterising the GAL1 promoter induction characteristics

Here, we successfully characterised the induction characteristics of the GAL1 promoter using construct GAL1-[GFP]

2. Switch characterisation

(a) Characterising the GAL1 promoter induction characteristics

Here, we successfully characterised the induction characteristics of the GAL1 promoter using construct GAL1p-[Npeptide-GFP]
Timed Induction of Gal1 Promoter using GAL1p-[Npep-GFP]

(b) Characterising the GAL1 promoter dose-responsiveness characteristics

Here, we successfully characterised the dose response characteristics of the GAL1 promoter using construct GAL1p-[Npeptide-GFP]
Galactose Dose Response of Gal1 Promoter using GAL1p-[Npep-GFP]

(c) Characterising the expression of MS2-CFP from the construct CUP1p-[MS2-CFP]

Here we identified the failure of the CUP1p-[MS2-CFP] construct to direct expression of the fusion protein at significant level, using a variety of analytical techniques to show that CFP expression was undetectable under a range of conditions

(d) Characterising the translational repression of GAL1p-[Npeptide-GFP] by trans expression of the MS2 protein.

Here, we used trans expression of the MS2 protein to show that the MS2 stem loops that formed part of the 5’ leader of the GAL1p-[Npeptide-GFP] mRNA were successfully recognised by the MS2 RNA binding protein, to cause translation repression of N-pep-GFP expression, validating our RNA stem loop-based translational control approach.
The effect of MS2 coat protein expresion on GAL1p-[Npep-GFP] expression
[[MS2 Coat-Protein Effect on Expression of GFP in pRS415]]

3. Switch troubleshooting

(a) Cassette replacement experiment – promoter

Descriptive Text....
Using homologous recombination to replace the CUP1 promoter in CUP1p-[MS2-CFP] with a CUP1 promoter plus 5' untranslated leader sequence

(b) Cassette replacement experiment – fluorescent protein

Descriptive Text....
Using homologous recombination to replace the CFP fluorescent protein in CUP1p-[MS2-CFP] with a GFP replacement variant

4. Other Biobrick testing

mOrange experiments

Homologous Recombination of E2050 into GAL1p-[Npep-GFP] Construct in Place of GFP Protein
FACS analysis of mOrange expression under Gal1 promoter control in GAL1p-[Npep-mOrange]