Team:Aberdeen Scotland/Constructs

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<h1>The DNA Constructs</h1>
<h1>The DNA Constructs</h1>
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<p>Throughout our project we used a variety of DNA constructs. Many of these have been
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closed and submitted into the <a href="link to Registry of Parts">Registry of Parts</a>.
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<p><b>The DNA constructs used in this project are organised into three main groups:</b></p>
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<ol>
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<LI>Promoter characterisation constructs <br>
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<LI>Switch components <br>
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<LI>Constructs for switch testing
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</ol>
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<p>
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<b><i>For each construct, we provide a brief description, and its intended use;</i></b>
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</p>
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<br><br>
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<h2>1. Promoter characterisation constructs</h2>
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<h4>GAL1p-[GFP]</h4>
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<hr>
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<p><b>Description:</b> This is a genomically-integrated construct in which the GFP protein was placed under control of the yeast GAL1 promoter (Fig. 1)
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</p><br>
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<p><b>Main use:</b> to characterise the induction characteristics of the <i>GAL1</i> promoter, which is induced by galactose. <a href="https://2010.igem.org/Team:Aberdeen_Scotland/Results"><i>See promoter activity assay results</i></a>
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</p>
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<br><h4>CUP1p-[GFP]</h4><hr>
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<p>
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<b>Description:</b> This is a genomically integrated construct in which the GFP protein was placed under control of the yeast CUP1 promoter.</p><br>
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<p><b>Main use:</b> to characterise the induction characteristics of the <i>CUP1</i> promoter, which is induced by copper ions. <a href="https://2010.igem.org/Team:Aberdeen_Scotland/Results"><i>See promoter activity assay results</i></a></p>
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<center><img src="https://static.igem.org/mediawiki/2010/e/ef/CUP1_promoter_and_GAL1_promoter.jpg"/></center>
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<br><br>
<br>
<br>
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The following is a description of the constructs that we have used and their functions.</p>
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<h2>2.Switch components</h2>
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<br><h4>CUP1p - [MS2-CFP]</h4><hr>
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<p><b>Description:</b> 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.</p><br>
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<p><b>Main use:</b> to be one half of the <b>AyeSwitch</b>. 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. </p>
<br>
<br>
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<h3>N4 and N5</h3>
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<p>N.B. It was discovered that this construct did not exhibit CFP fluorescence as expected when induced with Cu2+.<a href="https://2010.igem.org/Team:Aberdeen_Scotland/Results"><i>See 'Results'</i></a></p>
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<table>
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<tr>
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<br><br><br>
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<td>Constructs N4 and N5 as shown by (Fig.1) was designed an made by our advisor, Dr. I.
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<center><img src="https://static.igem.org/mediawiki/2010/f/f4/CUP1_promoter_and_Bbox_stem_loop.jpg"/></center>
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Stansfield, primarily to allow us to characterise the activity of the CUP1 and Gal1 promoters.
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<br><br><br>
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To do this, addition of the corresponding stimulating chemicals would activate the promoter
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and induce the production of GFP allowing the quantification of <a href="link to expts">promoter activity</a>.
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<h4>GAL1p-[Npep-GFP]</h4>
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for N4, the inducer was Copper (II) ions. (Cu2+).  For N5, the inducer was Galactose.</td>
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<td><img src="https://static.igem.org/mediawiki/2010/e/ef/CUP1_promoter_and_GAL1_promoter.jpg"/></td></tr></table>
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<p><b>Description:</b> 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.</p>
<br>
<br>
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<h3>pRS414</h3>
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<p>This is one of the constructs that makes up the <b>AyeSwitch</b> as shown by (Fig.2).
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<p><b>Main use;</b> to be one half of the <b>AyeSwitch</b>. 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. <a href="https://2010.igem.org/Team:Aberdeen_Scotland/Results"><i>See 'Results'</i></a></p>
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<center>
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<br><br><br>
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<img src="https://static.igem.org/mediawiki/2010/f/f4/CUP1_promoter_and_Bbox_stem_loop.jpg"/>
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<center><img src="https://static.igem.org/mediawiki/2010/9/9c/GAL1_promoter_and_MS2_Stem_Loops.jpg"/></center>
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</center>
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<br><br><br>
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The CUP 1 promoter in pRS414 is switched on by the addition of Cu(II) which initiates
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transcription.  The mRNA produced contains a Bbox stem loop that can be bound by N-peptide
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produced by pRS415.  This inhibits the translation of MS2-protein and cyan fluorescent
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<h4>Combining Gal1p-[Npep-GFP] and Cup1p-[MS2-CFP]: The <b>AyeSwitch</b></h4>
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protein, (CFP) which is coded downstream of Bbox stem loop in the pRS414 mRNA.
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<p>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.</p>
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<br><br><br>
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<center><img src="https://static.igem.org/mediawiki/2010/f/ff/Toggle_switch.jpg"/></center>
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<br><br><br>
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<h2>3.  Constructs for switch testing</h2>
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<br><h4>MET17p - [MS2]</h4>
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<p><b>Description:</b> 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 <i>MET17</i> promoter. This promoter is induced in the absence of methionine in the growth medium, and repressed by its presence.</p>
<br>
<br>
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In the absence of N-peptide, translation of MS2-protein and CFP occurs as usual.</p>
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<p><b>Main use;</b>  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.<a href="https://2010.igem.org/MS2_Coat-Protein_Effect_on_Expression_of_GFP_in_pRS415"><i>See results</i></a></p>
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<br><br><br>
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<center><img src="https://static.igem.org/mediawiki/2010/d/d0/MET17_promoter.jpg"/></center>
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<br><br><br>
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<h4>TEF1p -[CFP]</h4>
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<p><b>Description:</b> This construct consisted of a constitutively active TEF1 promoter controlling the expression of a CFP sequence downstream.</p>
<br>
<br>
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<h3>pRS415</h3>
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<p><b>Main use:</b> 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. <a href="https://2010.igem.org/Team:Aberdeen_Scotland/Results"><i>See 'Results/Troubleshooting'</i></a></p>
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<p>This is the other construct that makes up the <b>AyeSwitch</b> as shown by (Fig.3).
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<br>
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The Gal1 promoter in pRS415 is switched on by the addition of Galactose which initiates
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transcription.  The mRNA produced contains two MS2 stem loops that can be bound by MS2-protein
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produced by pRS414. This inhibits that translation of N-peptide and GFP which is coded downstream
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of the MS2 stem loops in the pRS415 mRNA.
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<br>
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In the absence of MS2-protein, translation of N-peptide and GFP occurs as usual.
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<br>
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<h3>The <b>AyeSwitch</b></h3>
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<p>Transforming yeast to contain both pRS414 and pRS415 creates the <b>AyeSwitch</b>.
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<br>
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From (Fig.4) it can be seen that there is mutual inhibition of pRS414 and pRS415
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at the translational level.  That is because the translated proteins of pRS414 and pRS415
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can bind to the corresponding stem loop structures on the opposing construct.
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<br>
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Binding of the stem loops by the correct protein inhibits the movement of ribosomes necessary
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for translation.  This prevents the syntheses of proteins coded downstream of the stem loops
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in the mRNA sequence.</p>
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<br><br><br>
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<center><img src="https://static.igem.org/mediawiki/2010/c/c7/TEF1_promoter.jpg"/></center>
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<br><br><br>
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<hr>
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<table class="nav">
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<td>
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<a href="https://2010.igem.org/Team:Aberdeen_Scotland/General_Overview"><img src="https://static.igem.org/mediawiki/2010/8/8e/Left_arrow.png">&nbsp;&nbsp;Return to Biology Summary</a>
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</td>
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<td align="right">
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<a href="https://2010.igem.org/Team:Aberdeen_Scotland/Results">Continue to Results Main Page&nbsp;&nbsp;<img src="https://static.igem.org/mediawiki/2010/3/36/Right_arrow.png"></a>
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{{:Team:Aberdeen_Scotland/Footer}}

Latest revision as of 09:34, 27 October 2010

University of Aberdeen - ayeSwitch - iGEM 2010

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'







  Return to Biology Summary Continue to Results Main Page  

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