Difference between revisions of "Team:SDU-Denmark/Tour31"

 
(127 intermediate revisions by 4 users not shown)
Line 3: Line 3:
  
  
<h1>Two-Hybrid Screening </h1>
+
<p> <i> "Coming together is a beginning; keeping together is progress; working together is success." - <b>Henry Ford</b></i></p>
<p>We are using a Two-Hybrid system to screen for aptameres as an alternate to antibodies. It can be used for detecting protein-protein interactions, by measuring levels of cAMP. The way a two-hybrid system functions is:
+
  
<a class="popupImg alignRight" style="width:100px" target="_blank" href="https://static.igem.org/mediawiki/2015/1/16/SDU2015_2-hybrid_screening.png" title="The peptide aptamer binds to the target resulting in a position of the two subunits of adenylate cyclase, close enough for its catalytic activity to function. The result is a conversion of ATP into cAMP.">
+
<h1 align="center">Two-Hybrid Screening </h1>
   <img src="https://static.igem.org/mediawiki/2015/1/16/SDU2015_2-hybrid_screening.png" style="width:200px"/>
+
<div class="thumb tright">
     <b>Figure 1:</b> T18 and T25 each contain one of the coding sequences for the two catalytic subunits of adenylate cyclase.
+
<div class="thumbinner" style="width:335px;height:140px;">
</a>
+
<a class="popupImg alignRight" style="width:330px" target="_blank" href="https://static.igem.org/mediawiki/2015/5/57/SDU2015_TwoHybridScreeningCyaA.png" title="Arrangement of adenylate cyclase toxin in <i>Bordetella pertussis</i>. The adenylate cyclase gene can be seperated into two fragments, T25 and T18.  Reconstitution of adenylate cyclase activity in a <i>cyaA<sup>-</sup></i> <i>E. coli</i> strain is the basis of the bacterial two hybrid system. Adapted from Battesti A. & Bouveret E. 2012">
 +
   <img src="https://static.igem.org/mediawiki/2015/f/f0/SDU2015_TwoHybridScreeningCyaA_thumbnail.png" style="width:330px"/></a>
 +
     <div class="thumbcaption">Figure 1: Arrangement of adenylate cyclase toxin gene in <i>Bordetella pertussi</i>.
 +
</div>
 +
</div>
 +
</div>
  
The subunits of the adenylate cyclase T18 and T25 functions only when close together. The linker ensures this. The cyclase is then able to convert ATP to cAMP. </p>
 
  
<h2>Our system </h2>
 
<p> In our Two-Hybrid screening system, the linker between T18 and T25 is;
 
A scaffold protein, coupled to T18, with the aptamer in its active site and on T25 a small linker domain followed by the target protein.
 
To detect an increase in cAMP levels, a MG1655:delta-CyA  is used. Detection is insured by a PstA a cAMP-activated promotor. The product of transcription is RFP (red fluorescent protein) visible by the eye and with fluoresces. The stronger the interaction between aptamer and target, the stronger the fluorescent. Selection of high affinity aptameres is thereby enabled.
 
  
(billedscreening system)
+
<p>
 +
<span class="intro">The bacterial two-hybrid system is a technology</span> used to detect protein-protein interactions. It is based on the <span class="tooltipLink">adenylate cyclase</span><span class="tooltip">
 +
<span class="tooltipHeader">Adenylate Cyclase</span>Adenylate cyclase is a transmembrane enzyme that generates the second messenger cyclic adenosine monophosphate (cAMP). This signalling pathway is activated in a low-energy metabolic state.</span>
 +
  activity reconstitution in a Δ<i>cyaA Eschericia coli</i> stra<span class="sourceReference">in</span>.
 +
<span class="tooltip">
 +
  <span class="tooltipHeader">Reference:</span>
 +
Karimova G, Pidoux J, Ullmann A, Ladant D. (1998) A bacterial two-hybrid system based on a reconstituted signal transduction pathway. 1998;95(10):5752-6.
 +
  <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC20451/ "> [PubMed] </a>
 +
<br>
 +
<br>
 +
Battesti A, Bouveret E. (2012) The bacterial two-hybrid system based on adenylate cyclase reconstitution in Escherichia coli. 2012;58(4):325-34. <br> DOI:10.1016/j.ymeth.2012.07.018
 +
  <a target="_blank" href="http://www.sciencedirect.com/science/article/pii/S1046202312001740 "> [ScienceDirect] </a>
 +
</span>
  
After selection, the specific sequence for an aptamer + linker and scaffold is cut out and transferred, to a another plasmid containing intein, used for production. This colony has the . system promoting secretion of our product. As part of the construct is the intein, which is self cleaving used for purification of product in an EBA. </p>
+
In this system the two proteins are generally referred to as "Bait" and "Pr<span class="sourceReference">ey"</span>.
 +
<span class="tooltip">
 +
  <span class="tooltipHeader">Reference:</span>
 +
Battesti A, Bouveret E. (2012) The bacterial two-hybrid system based on adenylate cyclase reconstitution in Escherichia coli. 2012;58(4):325-34. <br> DOI:10.1016/j.ymeth.2012.07.018
 +
  <a target="_blank" href="http://www.sciencedirect.com/science/article/pii/S1046202312001740 "> [ScienceDirect] </a>
 +
</span>
 +
 
 +
If there is an interaction between the two, it will lead to cAMP synthesis. This will trigger transcription of a reporter system that leads to a detectable phenotypic chan<span class="sourceReference">ge</span>.
 +
<span class="tooltip">
 +
  <span class="tooltipHeader">Reference:</span>
 +
Karimova G, Pidoux J, Ullmann A, Ladant D. (1998) A bacterial two-hybrid system based on a reconstituted signal transduction pathway. 1998;95(10):5752-6. <br>
 +
  <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC20451/ "> [PubMed] </a>
 +
</span>
 +
<br>
 +
We are using this technique to screen for functioning peptide aptamers that are able to bind our chosen target protein, thus functioning as an alternative to antibodies.
 +
</p>
 +
 
 +
<div class="thumb tright">
 +
<div class="thumbinner" style="width:405px;height:225px;">
 +
<a class="popupImg alignRight" style="width:400px" target="_blank" href="https://static.igem.org/mediawiki/2015/4/4c/SDU2015_T18andT25interaction.png" title="<b>A:</b> When <i>cyaA</i> is expressed in a <i>cyaA<sup>-</sup></i> <i>E. coli</i> strain, adenylate cyclase activity is reconstituted. <b>B:</b> When the two fragments T25 and T18 is expressed separetely in a <i>cyaA<sup>-</sup></i> <i>E. coli</i> strain, no cAMP will be produced. <b>C:</b> If T25 and T18 are linked to the interacting Bait and Prey proteins, the proximity of the domains restores adenylate cyclase-activity, enableling synthesis of cAMP. Adapted from Battesti A. & Bouveret E. 2012">
 +
  <img src="https://static.igem.org/mediawiki/2015/c/c2/SDU2015_T18andT25interaction_thumbnail.png" style="width:400px"/></a>
 +
    <div class="thumbcaption">Figure 2: Interacting proteins can be detected using the bacterial two-hybrid system</div>
 +
 
 +
</div>
 +
</div>
 +
 
 +
<p>
 +
<span class="intro">Generally, the bacterial two-hybrid system</span> exploits the catalytic activity of adenylate cyclase to generate cAMP. The system we use was first described 17 years ago by Kariomva et <span class="sourceReference">al.</span>.
 +
<span class="tooltip">
 +
  <span class="tooltipHeader">Reference:</span>
 +
Karimova G, Pidoux J, Ullmann A, Ladant D. (1998) A bacterial two-hybrid system based on a reconstituted signal transduction pathway. 1998;95(10):5752-6. <br>
 +
  <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC20451/ "> [PubMed] </a>
 +
</span>
 +
In the system the two domains of the adenylate cyclase toxin gene (<i>cyaA</i>) of <i>Bordetella pertussis</i>, called T18 and T25, are located on two different plasmids. The domains are linked to the nucleotide sequences of the two proteins of interest, generating so-called hybrid gen<span class="sourceReference">es</span>.
 +
<span class="tooltip">
 +
  <span class="tooltipHeader">Reference:</span>
 +
Karimova G, Pidoux J, Ullmann A, Ladant D. (1998) A bacterial two-hybrid system based on a reconstituted signal transduction pathway. 1998;95(10):5752-6. <br>
 +
  <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC20451/ "> [PubMed] </a>
 +
</span>
 +
 
 +
If the "Prey" protein is able to interact with the "Bait" protein, the two catalytic domains will be brought into close proximity, enabling synthesis of cAMP from ATP.
 +
cAMP will bind the Catabolite Activating Protein (CAP). The complex can induce expression of a various set of reporter-genes controlled by a cAMP/CAP-dependent promot<span class="sourceReference">er</span>.
 +
<span class="tooltip">
 +
  <span class="tooltipHeader">Reference:</span>
 +
Karimova G, Pidoux J, Ullmann A, Ladant D. (1998) A bacterial two-hybrid system based on a reconstituted signal transduction pathway. 1998;95(10):5752-6.
 +
  <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC20451/ "> [PubMed] </a>
 +
<br>
 +
<br>
 +
Battesti A, Bouveret E. (2012) The bacterial two-hybrid system based on adenylate cyclase reconstitution in Escherichia coli. 2012;58(4):325-34. <br> DOI:10.1016/j.ymeth.2012.07.018
 +
  <a target="_blank" href="http://www.sciencedirect.com/science/article/pii/S1046202312001740 "> [ScienceDirect] </a>
 +
</span>
 +
If you are interested in a different type of application of 'the bacterial two-hybrid system' click <a class="dialogLink" href="https://2015.igem.org/Team:SDU-Denmark/DialogLink1">here</a>.
 +
</p>
 +
 
 +
<div class="thumb tright">
 +
<div class="thumbinner" style="width:335px;height:140px;">
 +
<a class="popupImg alignRight" style="width:330px" target="_blank" href="https://static.igem.org/mediawiki/2015/2/2d/RFP-reporterSDU.jpeg" title="The PcstA-induced transcription and expression of Red Fluorescence Protein, leading to red colored bacteria.">
 +
  <img src="https://static.igem.org/mediawiki/2015/2/2d/RFP-reporterSDU.jpeg" style="width:330px"/></a>
 +
    <div class="thumbcaption">Figure 3: The RFP reporter system
 +
</div>
 +
</div>
 +
</div>
 +
 
 +
<p>
 +
<span class="intro">The reporter system</span> which we initially intended to use was a cAMP/CAP-dependent transcription of the gene encoding Red Fluorescent Protein (RFP). On the target plasmid, transcription of RFP was controlled by the cAMP/CAP-sensitive promoter PcstA <a href="http://parts.igem.org/Part:BBa_K118011" target="_blank">(BBa_K118011)</a>. In this reporter system protein-protein interactions would result in expression of red fluorescence.
 +
</p>
 +
 
 +
 
 +
<div class="thumb tright">
 +
<div class="thumbinner" style="width:405px;height:290px;">
 +
<a id="Figure3" class="popupImg alignRight" style="width:400px" target="_blank" href="https://static.igem.org/mediawiki/2015/f/f5/SDU2015_lacZandXgal.png" title="<u><b>The chromosomal X-gal/<i>lacZ</i> reporter system of bacterial strain BTH101</u></b>. </p> <p><b>A:</b> cAMP will co-activate along with CAP the transcription of the gene <i>lacZ</i>, which encodes the enzyme β-galactosidase. <p> <b>B</b>: β-galactosidase can hydrolyze 5-bromo-4-chloro-3-indolyl-β-D-galactopyranosid to 5-bromo-4-chloro-3-hydroxyindole. This intermediate will be oxidized, which causes dimerization and formation of 5,5'-dibromo-4,4'-dichloro indigo. This final product has a blue color. Thus <i>lacZ</i> transcription, induced by intracellular levels of cAMP, leads to formation of a blue color. ">
 +
  <img src="https://static.igem.org/mediawiki/2015/9/92/SDU2015_lacZandXgal_thumbnail.png" style="width:400px"/></a>
 +
    <div class="thumbcaption">Figure 4: The X-gal/<i>lacZ</i> reporter system</div>
 +
</div>
 +
</div>
 +
 
 +
<p>
 +
<span class="intro">
 +
Due to difficulties</span> using this promoter we changed to a different reporter system. We used the bacterial strain BHT101, which was adenylate cyclase-deficient and contained a chromosomal <i>lacZ</i>-reporter system. In this reporter system cAMP will induce transcription of the <i>lacZ</i> gene, which encodes the enzyme β-galactosidase. If the bacteria is grown on plates containing
 +
<span class="tooltipLink">X-gal</span><span class="tooltip">
 +
<span class="tooltipHeader">X-gal</span>X-gal is the common name of 5-bromo-4-chloro-3-indolyl-β-D-galactopyranosid, an analoge of the disaccharide lactose.</span>, β-galactosidase can hydrolyze X-gal to a blue colored substrate (consult <a href="#Figure3">Figure 4</a> for a schematic overview). Bacteria with this reporter system would become blue when the two proteins are able to interact with each other.
 +
</p>
 +
 
 +
<p>
 +
<span class="intro">The system is suitable for screening</span> of different libraries. The system has been used to determine interaction partners in genomic librari<span class="sourceReference">es</span>.
 +
<span class="tooltip">
 +
  <span class="tooltipHeader">Reference:</span>
 +
Battesti A, Bouveret E. (2012) The bacterial two-hybrid system based on adenylate cyclase reconstitution in Escherichia coli. 2012;58(4):325-34. <br> DOI:10.1016/j.ymeth.2012.07.018
 +
  <a target="_blank" href="http://www.sciencedirect.com/science/article/pii/S1046202312001740 "> [ScienceDirect] </a>
 +
</span>
 +
 
 +
Our aim is, however, to generate a new interaction partner to a target protein; a peptide aptamer. This should be done through screening of a randomly generated nucleotide library against a chosen target protein.
 +
</p>
  
 
</html>
 
</html>
  
 
{{:Team:SDU-Denmark/core/footer}}
 
{{:Team:SDU-Denmark/core/footer}}

Latest revision as of 16:54, 4 October 2015

"Coming together is a beginning; keeping together is progress; working together is success." - Henry Ford

Two-Hybrid Screening

Figure 1: Arrangement of adenylate cyclase toxin gene in Bordetella pertussi.

The bacterial two-hybrid system is a technology used to detect protein-protein interactions. It is based on the adenylate cyclase Adenylate CyclaseAdenylate cyclase is a transmembrane enzyme that generates the second messenger cyclic adenosine monophosphate (cAMP). This signalling pathway is activated in a low-energy metabolic state. activity reconstitution in a ΔcyaA Eschericia coli strain. Reference: Karimova G, Pidoux J, Ullmann A, Ladant D. (1998) A bacterial two-hybrid system based on a reconstituted signal transduction pathway. 1998;95(10):5752-6. [PubMed]

Battesti A, Bouveret E. (2012) The bacterial two-hybrid system based on adenylate cyclase reconstitution in Escherichia coli. 2012;58(4):325-34.
DOI:10.1016/j.ymeth.2012.07.018 [ScienceDirect]
In this system the two proteins are generally referred to as "Bait" and "Prey". Reference: Battesti A, Bouveret E. (2012) The bacterial two-hybrid system based on adenylate cyclase reconstitution in Escherichia coli. 2012;58(4):325-34.
DOI:10.1016/j.ymeth.2012.07.018 [ScienceDirect]
If there is an interaction between the two, it will lead to cAMP synthesis. This will trigger transcription of a reporter system that leads to a detectable phenotypic change. Reference: Karimova G, Pidoux J, Ullmann A, Ladant D. (1998) A bacterial two-hybrid system based on a reconstituted signal transduction pathway. 1998;95(10):5752-6.
[PubMed]

We are using this technique to screen for functioning peptide aptamers that are able to bind our chosen target protein, thus functioning as an alternative to antibodies.

Figure 2: Interacting proteins can be detected using the bacterial two-hybrid system

Generally, the bacterial two-hybrid system exploits the catalytic activity of adenylate cyclase to generate cAMP. The system we use was first described 17 years ago by Kariomva et al.. Reference: Karimova G, Pidoux J, Ullmann A, Ladant D. (1998) A bacterial two-hybrid system based on a reconstituted signal transduction pathway. 1998;95(10):5752-6.
[PubMed]
In the system the two domains of the adenylate cyclase toxin gene (cyaA) of Bordetella pertussis, called T18 and T25, are located on two different plasmids. The domains are linked to the nucleotide sequences of the two proteins of interest, generating so-called hybrid genes. Reference: Karimova G, Pidoux J, Ullmann A, Ladant D. (1998) A bacterial two-hybrid system based on a reconstituted signal transduction pathway. 1998;95(10):5752-6.
[PubMed]
If the "Prey" protein is able to interact with the "Bait" protein, the two catalytic domains will be brought into close proximity, enabling synthesis of cAMP from ATP. cAMP will bind the Catabolite Activating Protein (CAP). The complex can induce expression of a various set of reporter-genes controlled by a cAMP/CAP-dependent promoter. Reference: Karimova G, Pidoux J, Ullmann A, Ladant D. (1998) A bacterial two-hybrid system based on a reconstituted signal transduction pathway. 1998;95(10):5752-6. [PubMed]

Battesti A, Bouveret E. (2012) The bacterial two-hybrid system based on adenylate cyclase reconstitution in Escherichia coli. 2012;58(4):325-34.
DOI:10.1016/j.ymeth.2012.07.018 [ScienceDirect]
If you are interested in a different type of application of 'the bacterial two-hybrid system' click here.

Figure 3: The RFP reporter system

The reporter system which we initially intended to use was a cAMP/CAP-dependent transcription of the gene encoding Red Fluorescent Protein (RFP). On the target plasmid, transcription of RFP was controlled by the cAMP/CAP-sensitive promoter PcstA (BBa_K118011). In this reporter system protein-protein interactions would result in expression of red fluorescence.

Figure 4: The X-gal/lacZ reporter system

Due to difficulties using this promoter we changed to a different reporter system. We used the bacterial strain BHT101, which was adenylate cyclase-deficient and contained a chromosomal lacZ-reporter system. In this reporter system cAMP will induce transcription of the lacZ gene, which encodes the enzyme β-galactosidase. If the bacteria is grown on plates containing X-gal X-galX-gal is the common name of 5-bromo-4-chloro-3-indolyl-β-D-galactopyranosid, an analoge of the disaccharide lactose., β-galactosidase can hydrolyze X-gal to a blue colored substrate (consult Figure 4 for a schematic overview). Bacteria with this reporter system would become blue when the two proteins are able to interact with each other.

The system is suitable for screening of different libraries. The system has been used to determine interaction partners in genomic libraries. Reference: Battesti A, Bouveret E. (2012) The bacterial two-hybrid system based on adenylate cyclase reconstitution in Escherichia coli. 2012;58(4):325-34.
DOI:10.1016/j.ymeth.2012.07.018 [ScienceDirect]
Our aim is, however, to generate a new interaction partner to a target protein; a peptide aptamer. This should be done through screening of a randomly generated nucleotide library against a chosen target protein.