Difference between revisions of "Team:SDU-Denmark/Tour32"

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   <img src="https://static.igem.org/mediawiki/2015/2/24/Targets_SDU-Denmark.png" style="width:350px;"/>
 
   <img src="https://static.igem.org/mediawiki/2015/2/24/Targets_SDU-Denmark.png" style="width:350px;"/>
 
</a>
 
</a>
<div class="thumbcaption"><b>Figure 1:</b> Target Device: Target protein fused to T25 through a flexible linker. Device also contains a cAMP-induced RFP reporter system.</div>
+
<div class="thumbcaption"><i>Figure 1:</i> <b>Target Device:</b> Target protein fused to T25 through a flexible linker. Device also contains a cAMP-induced RFP reporter system.</div>
 
</div>
 
</div>
 
</div>
 
</div>
 
<p>
 
<p>
<span class="intro">The two-hybrid system is designed in 2 plasmids.</span> One plasmid containing the complex build around T18 and one plasmid with the T25-complex. The cells need to take up both plasmids. To be able to select for the cells which have obtain both plasmids need to have resistance for different antibiotics, in this case resistance for chloramphenicol (CML) and Kanamycin (Kan) and therefore the plasmid backbones used was standard iGEM backbones: pSB1C3 and pSB1K3 respectively.  
+
<span class="intro"> The bacterial two-hybrid screening system </span> is made up of two primary devices. One that is build around the T18 domain and one that is build around the T25 domain.
 +
<br><br>
 +
<span class="intro"> Our device with the T25 domain </span> is our target device. It contains a target conjugated to the T25 domain through a flexible linker. The gene is controlled by the <i>lac</i> promoter,
 +
<span class="tooltipLink">Plac</span><span class="tooltip">
 +
<span class="tooltipHeader">lac promoter</span>The <i>lac</i> promoter is inducible by isopropyl-beta-D-1-thiogalactopyranoside (IPTG). The promoter has two binding sites: one which can bind catabolite associated protein (CAP), that binds cyclin adenosinemonophosphate (cAMP) and another which can bind the repressor protein LacI. When IPTG binds LacI, the repressor protein is inhibited.
 +
</span>
 +
The <i>lac</i> promoter is chosen in this case because, while it is inducible, it also gives a high enough expression when expressed in high-copy plasmids such as pSB1C3 and pSB1K3.  
 
</p>
 
</p>
  
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</a>
 
</a>
<div class="thumbcaption"><b>Figure 2:</b> Peptide aptamer Device: Peptide aptamer-scaffold fused to the T18 domain through a flexible linker.</div>
+
<div class="thumbcaption"><i>Figure 2:</i> <b>Peptide aptamer Device:</b> Peptide aptamer-scaffold fused to the T18 domain through a flexible linker.</div>
 
</div>
 
</div>
 
</div>
 
</div>
 +
<p>
 +
<span class="intro"> This device</span> also contains a generator of red fluorescent protein (RFP) controlled by the <i>cstA</i> promoter. PcstA is a carbohydrate stress promoter and is induced by cAMP. This make it usable as a reporter system for our bacterial two-hybrid system that is based on the reconstitution of adenylate cyclase upon protein-protein interaction between the two proteins conjugated to T18 and T25. If using a <i>cyaA</i>-deficient strain, red colonies will only be seen when a protein-protein interaction occurs.
 +
</p>
 +
<p>
 +
In our project we worked with the three target proteins: carbon storage regulator CsrA (<a href="http://parts.igem.org/Part:BBa_K1638037">BBa_K1638037</a>), the RNase adaptive protein Yhbj (<a href="http://parts.igem.org/Part:BBa_K1638038">BBa_K1638038</a>) and the RNA-binding protein Hfq (<a href="http://parts.igem.org/Part:BBa_K1638039">BBa_K1638039</a>).
 +
</p>
  
 
<p>
 
<p>
<span class="intro">The brick containing T25 consists</span> of a PcstA promoter, a RBS and the gene encoding for red fluorescent protein (RFP) followed by two transcriptional terminators consisting each of a 64 bp stem-loop. Located downstream are a Plac promoter and RBS followed by T25 ligated to the gene of one of the 3 target proteins: carbon storage regulator A CsrA (<a href="http://parts.igem.org/Part:BBa_K1638037" target="_blank">BBa_K1638037</a>), the RNase adaptive protein Yhbj (<a href="http://parts.igem.org/Part:BBa_K1638038" target="_blank">BBa_K1638038</a>) and the RNA-binding protein Hfq (<a href="http://parts.igem.org/Part:BBa_K1638039" target="_blank">BBa_K1638039</a>).
+
<span class="intro"> Our device with T18 domain </span> is our peptide aptamer device. It contains the human thioredoxin (hTrx)-based peptide aptamer conjugated to the T18 domains through a flexible linker. The device is controlled by the strong hybrid promoter PLlac<span class="sourceReference">O-1</span>.
 +
<span class="tooltip">
 +
  <span class="tooltipHeader">Reference:</span>
 +
Lutz R, Bujard H. Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements. Nucleic Acids Res. 1997 Mar 15;25(6):1203-10 <br>
 +
  <a target="_blank" href=" http://www.ncbi.nlm.nih.gov/pmc/articles/PMC146584// "> [PubMed] </a>
 +
</span>
 +
High expression is essential for a high yield of our peptide aptamer.
 
</p>
 
</p>
 
 
 
<div class="thumb tright">
 
<div class="thumb tright">
 
<div class="thumbinner" style="width:365px; height:135px;">
 
<div class="thumbinner" style="width:365px; height:135px;">
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   <img src="https://static.igem.org/mediawiki/2015/2/21/T18LIS_SDU-Denmark.png" style="width:350px"/>
 
   <img src="https://static.igem.org/mediawiki/2015/2/21/T18LIS_SDU-Denmark.png" style="width:350px"/>
 
</a>
 
</a>
<div class="thumbcaption"><b>Figure 3:</b> Modified peptide aptamer Device: Peptide aptamer-scaffold fused to the T18 domain through intein and a flexible linker.</div>
+
<div class="thumbcaption"><i>Figure 3:</i> <b>Modified peptide aptamer Device:</b> Peptide aptamer-scaffold fused to the T18 domain through intein and a flexible linker.</div>
 
</div>
 
</div>
 
</div>
 
</div>
 
 
 
 
 
<p>
 
<p>
<span class="intro">The promoter Plac is induced</span> by isopropyl-beta-D-1-thiogalactopyranoside (IPTG). The promoter has two binding sites: one which can bind catabolite associated protein (CAP), which is associated with glucose uptake and another which can bind the repressor protein LacI. When IPTG binds LacI, the repressor protein is inhibited. The inducible Plac promoter is chosen in this case because it can be regulated by environmental factors, but leaky enough to give a constant transcription with little to no activat<span class="sourceReference">ion</span>.
+
<span class="intro"> The hTrx-scaffold </span>contains a xhoI restriction site that enables insertion of a random nucleotide library. The resctrion site is situated in the active site of hTrx leaving it functionless when a library is inserted. The design of this peptide aptamer is inspired by Borghouts C
 +
<span class="sourceReference">et al</span>.
 
<span class="tooltip">
 
<span class="tooltip">
 
   <span class="tooltipHeader">Reference:</span>
 
   <span class="tooltipHeader">Reference:</span>
Kay Terpe. - Overview of bacterial expression systems for heterologous protein production: from molecular and biochemical fundamentals to commercial systems. Received: 30 January 2006. Revised: 18 April 2006. Accepted: 19 April 2006. Published online: 22 June 2006. Springer-Verlag 2006
+
Borghouts C, Kunz C, Delis N, Groner B. Monomeric Recombinant Peptide Aptamers Are Required for Efficient Intracellular Uptake and Target Inhibition. Molecular Cancer Research. 2008;6(2):267-81. <br>
   <a target="_blank" href="http://link.springer.com/article/10.1007%2Fs00253-006-0465-8"> (Link) </a>
+
   <a target="_blank" href=" http://mcr.aacrjournals.org/content/6/2/267.long "> [Mol Canc Res] </a>
 
</span>
 
</span>
 +
They modified this scaffold for optimal use. This includes mutations that prevents multimerization of the protein and induces flexibility of the inserted peptide aptamer. See <a href="http://parts.igem.org/Part:BBa_K1638014">BBa_K1638014</a> for further details.
 
</p>
 
</p>
 +
<p>
 +
A 3xFLAG-tag is added to the C-terminal end of the scaffold. This affinity tag can be used for detection and/or purification purpo
 +
<span class="sourceReference">ses</span>.
 +
<span class="tooltip">
 +
  <span class="tooltipHeader">Reference:</span>
 +
Sigmar-Aldrich: 3xFLAG system. Visited: 16.09.15 - <br>
 +
  <a target="_blank" href=" http://www.sigmaaldrich.com/life-science/molecular-biology/cloning-and-expression/vector-systems/mat-system-vectors/3x-flag.html#Figure 1 "> </a>
 +
</span>
 +
</p>
 +
<p>
 +
<span class="intro"> We also made</span> this device so that it contained intein.
  
  
 +
<span class="tooltipLink">Intein</span><span class="tooltip">
 +
<span class="tooltipHeader">Intein</span> Intein is a thiol-induced self-cleavable protein that enables the release of a C-terminal fused protein. It also contains a chitin-binding domain, which is an affinity-tag that enables affinity purification on a chitin column. First the target protein fused to intein is loaded and washed on the chitin column. Using a thiol reagent, like dithiothreitol (DTT), an on-column cleavage is induced and the target protein is released.
 +
</span>
 +
should enable affinity purification of the  hTrx-based peptide aptamer.
 +
</p>
 
<p>
 
<p>
<span class="intro">The brick containing T18</span> (<a href="http://parts.igem.org/Part:BBa_K1638018" target="_blank">BBa_K1638018</a>) consists of a PLlacO-1 promoter, a ribosomal binding site (RBS), T18 domain fused to a linker, which again is fused with human thioredoxin (hTrx). The peptide library is located inside the hTrx, which is fused with 3xFLAG.  
+
<span class="intro">To validate</span> that the T18 and T25 domain constructs can be used to study protein-protein interactions, we fused leucine zippers to the two catalytic domains. Read more about this in our <a href="https://2015.igem.org/Team:SDU-Denmark/Tour52">control experiment</a>.
 
</p>
 
</p>
 
 
 
 
<div class="thumb tright">
 
<div class="thumb tright">
 
<div class="thumbinner" style="width:365px; height:170px;">
 
<div class="thumbinner" style="width:365px; height:170px;">
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   <img src="https://static.igem.org/mediawiki/2015/e/ef/TZip_SDU-Denmark.png" style="width:350px"/>
 
   <img src="https://static.igem.org/mediawiki/2015/e/ef/TZip_SDU-Denmark.png" style="width:350px"/>
 
</a>
 
</a>
<div class="thumbcaption"><b>Figure 4:</b> Control device: Leucine zippers fused to the T18 and T25 domains of the adenylate cyclase CyaA.</div>
+
<div class="thumbcaption"><i>Figure 4:</i> <b>Control device:</b> Leucine zippers fused to the T18 and T25 domains of the adenylate cyclase CyaA.</div>
 
</div>
 
</div>
 
</div>
 
</div>
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   <img src="https://static.igem.org/mediawiki/2015/6/6d/SDU2015_LeucinZipper_Proteins_thumbnail.png" style="width:140px"/>
 
   <img src="https://static.igem.org/mediawiki/2015/6/6d/SDU2015_LeucinZipper_Proteins_thumbnail.png" style="width:140px"/>
 
</a>
 
</a>
<div class="thumbcaption"><b>Figure 5:</b> Interaction between BBa_K1638030 and BBa_K1638031. </div>
+
<div class="thumbcaption"><i>Figure 5:</i> Interaction between BBa_K1638030 and BBa_K1638031. </div>
 
</div>
 
</div>
 
</div>
 
</div>
 
 
<p>
 
<span class="intro">The PLlacO-1 is a regulatory region</span> consisting of a promoter P(L) of phage lambda with the cI binding sites replaced with lacO1. This hybrid design induce strong promotion, which can be inhibited by LacI and induced by IPTG and L-arabinose, because both molecules inhibit LacI. This promoter has been chosen for this construct because it is a very strong promoter, but at the same time it is highly repressible, therefore PLlacO-1 would give a much greater yield compared to a normal Plac promo<span class="sourceReference">ter</span>.
 
<span class="tooltip">
 
  <span class="tooltipHeader">Reference:</span>
 
Lutz R, Bujard H. - Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements. Nucleic Acids Res. 1997 Mar 15;25(6):1203-10.
 
  <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC146584/pdf/251203.pdf"> [PubMed] </a>
 
</span>
 
 
3x-FLAG is an affinity tag, which can be used for detection or purification. Fr analysis of the aptamer through western blotting the 3xFLAG is good mainly because of the small size and effectiveness. 3xFLAG system is an effective system for detection of fusion proteins and because of the small size there is a low risk of altering the aptamers function and struct<span class="sourceReference">ure</span>.
 
<span class="tooltip">
 
  <span class="tooltipHeader">Reference:</span>
 
Sigmar-Aldrich: 3xFLAG system. Visited: 16.09.15 -
 
  <a target="_blank" href="http://www.sigmaaldrich.com/life-science/molecular-biology/cloning-and-expression/vector-systems/mat-system-vectors/3x-flag.html#Figure 1"> (Link) </a>
 
</span>
 
</p>
 
 
 
 
 
 
<p>
 
<span class="intro">For the purpose of making the peptide aptamer</span> more accessible for purification and western blotting we made a modification to the brick containing T18, hTrx and the library. Upstream from hTrx the protein intein was inserted into the plasmid (<a href="http://parts.igem.org/Part:BBa_K1638035" target="_blank">BBa_K1638035</a>). Intein is a thiol-induced self-cleavable protein, which can enables the release of a given protein fused to the C.terminal end of intein.
 
</p>
 
 
 
<p>
 
<span class="intro">Intein is because of its self-cleaving ability</span> a good addition when the peptide aptamer needs to be separated from the catalytic domain T18. This makes purification and using the aptamer much easier.
 
</p>
 
 
<p>
 
To test the design of the two-hybrid system we fused both T18 and T25 to leucine zippers (LeuZ) (<a href="http://parts.igem.org/Part:BBa_K1638030" target="_blank">BBa_K1638030</a> and <a href="http://parts.igem.org/Part:BBa_K1638031" target="_blank">BBa_K1638031</a>).
 
<p>
 
 
 
</p>
 
<span class="intro">These proteins are known to dimerize,</span> which in this case suits the purpose of testing the system well. The LeuZ’s and the two catalytic domains are fused together with a linker, which enables the possibility that T18 and T25 can interact due to the dimerization of the LeuZs.
 
</p>
 
  
  

Revision as of 22:32, 18 September 2015

"Design is everything. EVERYTHING!" - Paul Rand

System Design

Figure 1: Target Device: Target protein fused to T25 through a flexible linker. Device also contains a cAMP-induced RFP reporter system.

The bacterial two-hybrid screening system is made up of two primary devices. One that is build around the T18 domain and one that is build around the T25 domain.

Our device with the T25 domain is our target device. It contains a target conjugated to the T25 domain through a flexible linker. The gene is controlled by the lac promoter, Plac lac promoterThe lac promoter is inducible by isopropyl-beta-D-1-thiogalactopyranoside (IPTG). The promoter has two binding sites: one which can bind catabolite associated protein (CAP), that binds cyclin adenosinemonophosphate (cAMP) and another which can bind the repressor protein LacI. When IPTG binds LacI, the repressor protein is inhibited. The lac promoter is chosen in this case because, while it is inducible, it also gives a high enough expression when expressed in high-copy plasmids such as pSB1C3 and pSB1K3.

Figure 2: Peptide aptamer Device: Peptide aptamer-scaffold fused to the T18 domain through a flexible linker.

This device also contains a generator of red fluorescent protein (RFP) controlled by the cstA promoter. PcstA is a carbohydrate stress promoter and is induced by cAMP. This make it usable as a reporter system for our bacterial two-hybrid system that is based on the reconstitution of adenylate cyclase upon protein-protein interaction between the two proteins conjugated to T18 and T25. If using a cyaA-deficient strain, red colonies will only be seen when a protein-protein interaction occurs.

In our project we worked with the three target proteins: carbon storage regulator CsrA (BBa_K1638037), the RNase adaptive protein Yhbj (BBa_K1638038) and the RNA-binding protein Hfq (BBa_K1638039).

Our device with T18 domain is our peptide aptamer device. It contains the human thioredoxin (hTrx)-based peptide aptamer conjugated to the T18 domains through a flexible linker. The device is controlled by the strong hybrid promoter PLlacO-1. Reference: Lutz R, Bujard H. Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements. Nucleic Acids Res. 1997 Mar 15;25(6):1203-10
[PubMed]
High expression is essential for a high yield of our peptide aptamer.

Figure 3: Modified peptide aptamer Device: Peptide aptamer-scaffold fused to the T18 domain through intein and a flexible linker.

The hTrx-scaffold contains a xhoI restriction site that enables insertion of a random nucleotide library. The resctrion site is situated in the active site of hTrx leaving it functionless when a library is inserted. The design of this peptide aptamer is inspired by Borghouts C et al. Reference: Borghouts C, Kunz C, Delis N, Groner B. Monomeric Recombinant Peptide Aptamers Are Required for Efficient Intracellular Uptake and Target Inhibition. Molecular Cancer Research. 2008;6(2):267-81.
[Mol Canc Res]
They modified this scaffold for optimal use. This includes mutations that prevents multimerization of the protein and induces flexibility of the inserted peptide aptamer. See BBa_K1638014 for further details.

A 3xFLAG-tag is added to the C-terminal end of the scaffold. This affinity tag can be used for detection and/or purification purpo ses. Reference: Sigmar-Aldrich: 3xFLAG system. Visited: 16.09.15 -

We also made this device so that it contained intein. Intein Intein Intein is a thiol-induced self-cleavable protein that enables the release of a C-terminal fused protein. It also contains a chitin-binding domain, which is an affinity-tag that enables affinity purification on a chitin column. First the target protein fused to intein is loaded and washed on the chitin column. Using a thiol reagent, like dithiothreitol (DTT), an on-column cleavage is induced and the target protein is released. should enable affinity purification of the hTrx-based peptide aptamer.

To validate that the T18 and T25 domain constructs can be used to study protein-protein interactions, we fused leucine zippers to the two catalytic domains. Read more about this in our control experiment.

Figure 4: Control device: Leucine zippers fused to the T18 and T25 domains of the adenylate cyclase CyaA.
Figure 5: Interaction between BBa_K1638030 and BBa_K1638031.