Difference between revisions of "Team:SDU-Denmark/Tour32"
Jpettersen (Talk | contribs) |
|||
Line 10: | Line 10: | ||
<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">< | + | <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 | + | <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> | ||
Line 23: | Line 29: | ||
</a> | </a> | ||
− | <div class="thumbcaption">< | + | <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"> | + | <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;"> | ||
Line 37: | Line 53: | ||
<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">< | + | <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 | + | <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> | ||
− | + | 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:// | + | <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"> | + | <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;"> | ||
Line 65: | Line 92: | ||
<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">< | + | <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> | ||
Line 74: | Line 101: | ||
<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">< | + | <div class="thumbcaption"><i>Figure 5:</i> Interaction between BBa_K1638030 and BBa_K1638031. </div> |
</div> | </div> | ||
</div> | </div> | ||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
Revision as of 22:32, 18 September 2015
"Design is everything. EVERYTHING!" - Paul Rand
System Design
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.
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.
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.