Team:Aberdeen Scotland/Results

From 2010.igem.org

(Difference between revisions)
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<html>
<html>
<h1>Main Experimental Results</h1>
<h1>Main Experimental Results</h1>
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<h2>1.  Promoter characterisation</h2>
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<h4>(a) Characterising the CUP1 promoter induction characteristics</h4>
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Here, we successfully characterised the induction characteristics of the CUP1 promoter using construct CUP1-[GFP]
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[[Timed Induction of the CUP1 Promoter Using N4]]<br>
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[[Copper Dose Response of the CUP1 Promoter Using N4]]<br>
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<br><br>
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<h4> b) Characterising the GAL1 promoter induction characteristics  </h4>
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Here, we successfully characterised the induction characteristics of the GAL1 promoter using construct GAL1-[GFP]
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<br><br>
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<h2>2.  Switch characterisation</h2>
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<h4> (a) Characterising the GAL1 promoter induction characteristics </h4>
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Here, we successfully characterised the induction characteristics of the GAL1 promoter using construct GAL1p-[Npeptide-GFP]
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[[Timed Induction of Gal1 Promoter in pRS415]]<br>
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<br><br>
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<h4>  (b) Characterising the GAL1 promoter dose-responsiveness characteristics  </h4>
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Here, we successfully characterised the dose response characteristics of the GAL1 promoter using construct GAL1p-[Npeptide-GFP]
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[[Galactose Dose Response of Gal1 Promoter in pRS415]]<br>
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<br><br>
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<h4>  (c) Characterising the expression of MS2-CFP from the construct CUP1p-[MS2-CFP]</h4>
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Here we identified the failure of the CUP1p-[MS2-CFP] construct to direct expression of the fusion protein at significant level, showing using a variety of analyticla techniques that CFP expression was undetectable under a range of conditions
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<br><br>
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<h4>  (d) Characterising the translational repression of GAL1p-[Npeptide-GFP] by trans expression of the MS2 protein.</h4>
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Here, we used <i>trans</i> 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, validating our RNA control approach. <br>
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[[MS2 Coat-Protein Effect on Expression of GFP in pRS415]]<br>
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<br><br>
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<h2>3.  Switch troubleshooting</h2>
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<h4> (a) Cassette replacement experiment – promoter </h4>
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Text....
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<br><br>
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<h4>  (b) Cassette replacement experiment – fluorescent protein </h4>
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Text....
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<br><br>
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<h2>4. Other Biobrick testing </h2>
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<h4>  mOrange experiments </h4>
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[[Homologous Recombination of  E2050 into pRS415 Construct in Place of GFP Protein]]<br>
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[[FACS Analysis of mOrange recombinant pRS415]]<br>
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<br><br>
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<p>
<p>
<b>Characterising the pRS414 related components of the switch</b><br>
<b>Characterising the pRS414 related components of the switch</b><br>
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[[Galactose Dose Response of Gal1 Promoter in pRS415]]<br>
[[Galactose Dose Response of Gal1 Promoter in pRS415]]<br>
[[Timed Induction of Gal1 Promoter in pRS415]]<br>
[[Timed Induction of Gal1 Promoter in pRS415]]<br>
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<br>
+
<br><br>
<b>Characterising the mutual inhibition within the switch</b><br>
<b>Characterising the mutual inhibition within the switch</b><br>
[[MS2 Coat-Protein Effect on Expression of GFP in pRS415]]<br>
[[MS2 Coat-Protein Effect on Expression of GFP in pRS415]]<br>

Revision as of 16:10, 17 October 2010

University of Aberdeen - ayeSwitch - iGEM 2010

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 N4]]
[[Copper Dose Response of the CUP1 Promoter Using N4]]


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 in pRS415]]


(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 in pRS415]]


(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, showing using a variety of analyticla techniques 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, validating our RNA control approach.
[[MS2 Coat-Protein Effect on Expression of GFP in pRS415]]


3. Switch troubleshooting

(a) Cassette replacement experiment – promoter

Text....

(b) Cassette replacement experiment – fluorescent protein

Text....

4. Other Biobrick testing

mOrange experiments

[[Homologous Recombination of E2050 into pRS415 Construct in Place of GFP Protein]]
[[FACS Analysis of mOrange recombinant pRS415]]


Characterising the pRS414 related components of the switch
Timed Induction of the CUP1 Promoter Using N4
Copper Dose Response of the CUP1 Promoter Using N4


Characterising the pRS415 related components of the switch
Galactose Dose Response of Gal1 Promoter in pRS415
Timed Induction of Gal1 Promoter in pRS415


Characterising the mutual inhibition within the switch
MS2 Coat-Protein Effect on Expression of GFP in pRS415

Troubleshooting pRS414
Experimental Layout

Characterising Biobrick E2050 mOrange Fluorescent Protein
Homologous Recombination of E2050 into pRS415 Construct in Place of GFP Protein
FACS Analysis of mOrange recombinant pRS415

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