The DNA Constructs

The DNA constructs used in this project are organised into three main groups:

1. Promoter characterisation constructs
   
2. Switch components
   
3. Constructs for switch testing

For each construct, we provide a brief description, and its intended use;

1. Promoter characterisation constructs

GAL1p-[GFP]

Description: This is a genomically-integrated construct in which the GFP protein was placed under control of the yeast GAL1 promoter (Fig. 1)

Main use: to characterise the induction characteristics of the GAL1 promoter, which is induced by galactose. See promoter activity assay results

CUP1p-[GFP]

Description: This is a genomically integrated construct in which the GFP protein was placed under control of the yeast CUP1 promoter.

Main use: to characterise the induction characteristics of the CUP1 promoter, which is induced by copper ions. See promoter activity assay results

2.Switch components

CUP1p - [MS2-CFP]

Description: This construct was designed to be regulated by yeast CUP1 promoter. Downstream to this is a Bbox mRNA sequence followed by a fusion protein consisting of MS2 coat binding protein and CFP.

Main use: to be one half of the AyeSwitch. The Bbox mRNA sequence can be bound by N-peptide, (from GAL1p-Npep-GFP) which inhibits translation of MS2 coat binding protein and CFP. Translation of the MS2 coat binding protein allows inhibition of translation of Npep-GFP whilst CFP provides a means of quantification.

N.B. It was discovered that this construct did not exhibit CFP fluorescence as expected when induced with Cu2+.See 'Results'

GAL1p-[Npep-GFP]

Description: This construct was designed to be regulated by yeast GAL1 promoter. Downstream of this is a MS2 mRNA sequence followed by a fusion protein consisting of two N-peptide binding proteins and GFP.

Main use; to be one half of the AyeSwitch. The MS2 mRNA sequence can be bound by MS2 coat binding protein (from CUP1p-[MS2-CFP]) which inhibitions translation of N-peptide binding protein and GFP. Translation of the N-peptide binding protein allows inhibition of translation of MS2-CFP whilst GFP provides a means of quantification. See 'Results'

Combining Gal1p-[Npep-GFP] and Cup1p-[MS2-CFP]: The AyeSwitch

From the figure immediately below, it can be seen that there the Gal1p-[Npep-GFP] and Cup1p-[MS2-CFP] constructs are mutually inhibiting at the translational level. This is because the Npeptide, as part of an Npep-GFP fusion protein, is able to specifically bind the B-box mRNA stem loop in the Cup1p-[MS2-CFP] mRNA, and conversely, the MS2 protein, as part of an MS2-CFP fusion protein, is able to specifically bind the MS2 mRNA stem loops in the Gal1p-[Npep-GFP] mRNA. Each construct can be controlled transcriptionally using, respectively, galactose or copper ions in the growth medium.

3. Constructs for switch testing

MET17p - [MS2]

Description: This was a construct already avalilable in the host laboratory, in which the MS2 RNA-binding protein was placed under the control of an inducible MET17 promoter. This promoter is induced in the absence of methionine in the growth medium, and repressed by its presence.

Main use; The MS2 RNA binding protein binds MS2 RNA stem loops, such as those present in the GAL1p-[Npep-GFP] construct (see 'Switch components' above). Thus co-transforming GAL1p-[Npep-GFP] with MET17p - [MS2] in yeast would allow us to verify that MS2 protein binding to MS2 RNA stem loops would inhibit expression of N-pep-GFP at the translational level.See results

TEF1p -[CFP]

Description: This construct consisted of a constitutively active TEF1 promoter controlling the expression of a CFP sequence downstream.

Main use: for troubleshooting experiments. This construct was made by the team to allow us to verify that our fluorescent microscopes and cell cytometer (FACS) was able to successfully detect CFP expressed in yeast. It was used to confirm whether or not the CFP sequence from CUP1p - [MS2-CFP] were able to exhibit CFP fluorescence. See 'Results/Troubleshooting'

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