The DNA Constructs

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>> 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 Main use; to characterise the induction characteristics of the GAL1 promoter, which is induced by galactose. (Link to GAL1p-[GFP] characterisation experiments) 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. (Link to CUP1p-[GFP] characterisation experiments) 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+. 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. (Link to GAL1p -[Npep-GFP] characterisation experiments) 3. Constructs for switch testing MET17p - [MS2] Description: This was a Main use; to ...... TEF1p -[CFP] Description: This construct consisted of a constitutively TEF1 promoter with a CFP sequence downstream. Main use; for troubleshooting by checking the CFP sequence from CUP1p - [MS2-CFP] was able to exhibit CFP fluorescence. (Link to troubleshooting experiments) >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

Throughout our project we used a variety of DNA constructs. Many of these have been cloned and submitted into the Registry of Parts.
The following is a description of the constructs that we have used and their functions.

N4 and N5

Constructs N4 and N5 as shown by (Fig.1) was designed and made by our advisor, Dr. I. Stansfield, primarily to allow us to characterise the activity of the CUP1 and Gal1 promoters. To do this, addition of the corresponding stimulating chemicals would activate the promoter and induce the production of GFP allowing the quantification of promoter activity. for N4, the inducer was Copper (II) ions. (Cu2+). For N5, the inducer was Galactose.

pRS414

This is one of the constructs that makes up the AyeSwitch as shown by (Fig.2).

The CUP 1 promoter in pRS414 is switched on by the addition of Cu(II) which initiates transcription. The mRNA produced contains a Bbox stem loop that can be bound by N-peptide produced by pRS415. This inhibits the translation of MS2-protein and cyan fluorescent protein, (CFP) which is coded downstream of Bbox stem loop in the pRS414 mRNA. In the absence of N-peptide, translation of MS2-protein and CFP occurs as usual.

pRS415

This is the other construct that makes up the AyeSwitch as shown by (Fig.3).

The Gal1 promoter in pRS415 is switched on by the addition of Galactose which initiates transcription. The mRNA produced contains two MS2 stem loops that can be bound by MS2-protein produced by pRS414. This inhibits that translation of N-peptide and GFP which is coded downstream of the MS2 stem loops in the pRS415 mRNA. In the absence of MS2-protein, translation of N-peptide and GFP occurs as usual.

The AyeSwitch

Transforming yeast to contain both pRS414 and pRS415 creates the AyeSwitch. From (Fig.4) it can be seen that there is mutual inhibition of pRS414 and pRS415 at the translational level. This is because the translated proteins of pRS414 and pRS415 can bind to the corresponding stem loop structures on the opposing construct.

Binding of the stem loops by the correct protein inhibits the movement of ribosomes necessary for translation. This prevents the syntheses of proteins coded downstream of the stem loops in the mRNA sequence.

pMS2
We have used the MET17 promoter to characterise the inhibition of GFP expression caused by the MS2 protein binding to the MS2 loops.
However, with more time we would change this to produce CFP as a replacement for the pRS414 construct.

Ycp lac 22 Fl + CFP
This construct was used during testing to confirm that the CFP sequence being used was functional.

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