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

 
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<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 is the basis of the bacterial two hybrid system">
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<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>
 
   <img src="https://static.igem.org/mediawiki/2015/f/f0/SDU2015_TwoHybridScreeningCyaA_thumbnail.png" style="width:330px"/></a>
     <div class="thumbcaption"><i>Figure 1:</i> Arrangement of adenylate cyclase toxin gene in <i>Bordetella pertussi</i>.
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     <div class="thumbcaption">Figure 1: Arrangement of adenylate cyclase toxin gene in <i>Bordetella pertussi</i>.
 
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<span class="intro">The bacterial two-hybrid system is a technology</span> used to detect protein-protein interactions. It is based on <span class="tooltipLink">adenylate cyclase</span><span class="tooltip">
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<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 will generate the potent second messenger cyclic adenosine monophosphate (cAMP). This signalling pathway is activated in a low-energy metabolic state.</span>
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<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>.  
 
  activity reconstitution in a Δ<i>cyaA Eschericia coli</i> stra<span class="sourceReference">in</span>.  
 
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<a class="popupImg alignRight" style="width:400px" target="_blank" href="https://static.igem.org/mediawiki/2015/4/4c/SDU2015_T18andT25interaction.png" title="A: When <i>cyaA</i> is expressed in a <i>cyaA<sup>-</sup></i> <i>E. coli</i> strain, adenylate cyclase activity is reconstituted. 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. C: 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">
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<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>
 
   <img src="https://static.igem.org/mediawiki/2015/c/c2/SDU2015_T18andT25interaction_thumbnail.png" style="width:400px"/></a>
     <div class="thumbcaption"><i>Figure 2:</i> Interacting proteins can be detected using the bacterial two-hybrid system</div>
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     <div class="thumbcaption">Figure 2: Interacting proteins can be detected using the bacterial two-hybrid system</div>
  
 
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<span class="intro">Cenerally, 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>.
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<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="tooltip">
 
   <span class="tooltipHeader">Reference:</span>
 
   <span class="tooltipHeader">Reference:</span>
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   <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC20451/ "> [PubMed] </a>
 
   <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC20451/ "> [PubMed] </a>
 
</span>
 
</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 placed on two different plasmids. The domains are linked to the nucleotide sequence of the two proteins of interest, generating so-called hybrid gen<span class="sourceReference">es</span>.
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  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="tooltip">
 
   <span class="tooltipHeader">Reference:</span>
 
   <span class="tooltipHeader">Reference:</span>
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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.
 
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 to 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>.
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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>.
 
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   <span class="tooltipHeader">Reference:</span>
 
   <span class="tooltipHeader">Reference:</span>
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<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.">
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  <img src="https://static.igem.org/mediawiki/2015/2/2d/RFP-reporterSDU.jpeg" style="width:330px"/></a>
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    <div class="thumbcaption">Figure 3: The RFP reporter system
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<p>  
 
<p>  
<span class="intro">Before we could use the bacterial two-hybrid system </span> to screen for functioning peptide aptamers, we needed to verify the system, that it could indeed detect protein-protein interactions.<br>
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<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.
To this purpose we chose two proteins that were known to interact and linked them to T18 and T25. For this positive control we used two leucine zippers (LeuZ) which are known to form homodimers.
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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-sensitive promoter PcstA <a href="http://parts.igem.org/Part:BBa_K118011" target="_blank">(BBa_K118011)</a>. Bacteria with the combination T18-LeuZ and T25-LeuZ (and potentially functioning peptide aptamers) should turn red.
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<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. <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. ">
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<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>
 
   <img src="https://static.igem.org/mediawiki/2015/9/92/SDU2015_lacZandXgal_thumbnail.png" style="width:400px"/></a>
     <div class="thumbcaption"><i>Figure 3:</i> The X-gal/<i>lacZ</i> reporter system</div>
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     <div class="thumbcaption">Figure 4: The X-gal/<i>lacZ</i> reporter system</div>
 
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<span class="intro">
 
<span class="intro">
Due to</span> 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  
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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="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 3</a> for a schematic overview). Bacteria with this reporter system would become blue when the two proteins are able to interact with each other.
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<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.
 
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Our aim is, however, to generate a new interaction partner to a target protein; a peptide aptamer. We would do so by ordering a randomly generated nucleotide library. In the library every molecule should contain a different sequence of 60 base pairs. The sequence ordered was 3’-(…)-NNK-NNK-(…)-5’, meaning every third base should be either a guanine or cytosine. This should lower the risk of generating a stop-codon in the library. With this restriction it would mean that we would be able to generate
+
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.
 
+
<b>4<sup>40</sup> · 2<sup>20</sup> = 1,27 · 10<sup>30</sup> </b>
+
different nucleotide sequences.
+
Due to the degeneracy of the genetic code, however, this does not equal the possible peptide sequences that could be generated. If we temporary ignore the possibility of generating a stop codon and the degeneracy of the genetic code, this would mean that we could be able to generate
+
<b>20<sup>20</sup> = 1,05 · 10<sup>26</sup>
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</b>
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different amino acid sequences. It is very likely that somewhere in our library a nucleotide sequence would give rise to a functioning peptide aptamer that is able to bind our target protein.
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</p>
 
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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.