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]

Timed Induction of Gal1 Promoter using GAL1p-[Npep-GFP]

Galactose Dose Response of Gal1 Promoter using GAL1p-[Npep-GFP]

2.Switch characterisation

(a) Characterising the GAL1 promoter induction characteristics

In these experiments, 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) Measuring 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) Quantifying the expression of MS2-CFP from the construct CUP1p-[MS2-CFP]

This experiment 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

Confirmation that CUP1p-[MS2-CFP] did not express CFP

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

In these experiments, 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

(e) Measuring the decay characteristics of GFP following a switch to non-inducing conditions using GAL1p-[Npeptide-GFP].

In these experiments, a culture grown on galactose, and expressing GFP, was switched to glucose to repress GFP expresion, and the decay profile of GFP measured. The experiment revealed that GFP was extremely stable, and decay was primarily due to dilution through culture growth

The decay characteristics of GFP following promoter switch-off

3.Switch troubleshooting

(a) Cassette replacement experiment – promoter

Here we used homologous recombination to replace the CUP1 promoter in CUP1p-[MS2-CFP] with a previoulsy tested and functioning CUP1 promoter with 5' untranslated leader sequence CUP1 Characterisation in CUP1p-GFP and determined that the promoter was not the faulty component in CUP1p-[MS2-CFP].

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

Here we replaced the GFP sequence in TEF1p -[GFP] which constitutively expresses GFP with the CFP sequence from CUP1p-[MS2-CFP] and determined that the CFP sequence was expressed properly and therefore functioning correctly.

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

4. Other Biobrick testing

mOrange experiments

In these experiments, we tested the Biobrick E2050 mOrange from the Registry of Parts and confirmed that within our gene cassette,GAL1p-[Npep-GFP]this Biobrick part did not function as expected.

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]

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