Difference between revisions of "Team:SDU-Denmark/Tour73"

 
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<h1 align="center"> The end </h1>
 
  
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<p> <i> "The future belongs to those who believe in the beauty of their dreams." - <b>Eleanor Roosevelt</b></i></p>
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<h1 align="center"> Future Laboratory  Work </h1>
  
 
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<a class="popupImg alignRight" style="width:250px" target="_blank" href="https://static.igem.org/mediawiki/2015/b/b5/SDU2015_TheEndPhilosophy.jpg" title="Wanderer above the Sea of Fog (Casper David Friedrich in 1818)">
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<span class="intro">After a screen with the nucleotide library </span> and finding a peptide aptamer against a target protein the next step would be to examine the features of it.
  <img src="https://static.igem.org/mediawiki/2015/b/b5/SDU2015_TheEndPhilosophy.jpg" style="width:250px"/>  
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<span class="intro">The peptide aptamers affinity and specificity </span> towards its target are two important parameters to know, especially if it is meant for use in diagnostics and treatment.
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<span class="intro">The affinity is especially important </span> if we should get more than one positive colony in a screen. Several ways exist, to where we can test this, here amongs competitive Enzyme-Linked Immunosorbent Assay (ELISA). The ELISA wells would be coated with the target protein, to this we will add the peptide aptamer, giving them time to bind. After binding we will wash out any unbound peptide aptamers. A detection molecule that binds to the peptide aptamer though anti-FLAG will be added and later excess detection molecule will be washed out. An enzyme that converts the detections molecule to a color will be added. The color change or the amount of light emitted is proportional to the level of peptide aptamer bound to tar<span class="sourceReference">get</span>.
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<span class="tooltip">
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  <span class="tooltipHeader">Reference:</span>
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    Technical Guide for ELISA. Available from KPL, Kirkegaard & Perry Laboratories. <a target="blank_" href="http://www.kpl.com/docs/techdocs/KPL%20ELISA%20Technical%20Guide.pdf">(Link)
 
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</a>
<span class="intro">So now that you have made it to the very end</span> of our wiki site, we really want to thank you for taking the time to explore our project. Hopefully we have made it sound as interesting as we think it is, even though that is most unlikely.  
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Accessed 17 September 2015
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Thus the amount of peptide aptamer bound to target protein, provides a measurement for the affinity.
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The ELISA is competitive, thus the amount of target is realavlively small compared to that of peptide aptamers, this means the peptide aptamers will have to “compete” for binding the target protein.
 
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<span class="intro"> First step is the Giant Jamboree</span> on the 24<sup>th</sup>-28<sup>th</sup> of September in Boston, which we could not be more excited about. We are looking so much forward to meet all the other fantastic teams, some of them for the first time and some of them again. And not at least discover all the incredible possibilities of synthetic biology. </p>
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<a id="Figure1" class="popupImg alignRight" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2015/a/ad/Detector.png">
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  <img src="https://static.igem.org/mediawiki/2015/a/ad/Detector.png" style="width:180px"/></a>
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<div class="thumbcaption">Figure 1: Illustrates the detector with the Sensor chip, as the aqueous solution containing the target proteins flows by.
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<span class="intro">What are we thinking of when we think of the future?</span>
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<span class="intro">Surface Plasmon Resonance (SPR) </span> is a technology designed by Biacore and could be used to investigate the affinity and the specificity of the peptide aptamer.  Central for the SPR method is the movable sensor chip. This chip monitors the interaction between the peptide aptamer and its target. The operation of the instrument, data and the analysis of the data will be handled by a software.
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The sensor chip consist of a glass surface covered with a thin layer of gold, which forms a good basis to optimize the binding of a variety of molecules. To this chip the peptide aptamer would be bound.
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The target proteins will pass over the surface of the chip in a continuous, pulse-free and controlled flow, maintaining constant target protein concentration at the sensor chip surface. 
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SPR measures changes in refractive index, thus senses changes in mass in the aqueous layer close to the sensor c<span class="sourceReference">hip</span>.
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<span class="tooltip">
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  <span class="tooltipHeader">Reference:</span>
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    An Introduction to BiaCore's SPR Technology. Available from BiaCore.com <a target="blank_" href="http://www.rci.rutgers.edu/~longhu/Biacore/pdf_files/SPR_Technology_Brochure.pdf">(Link)
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Accessed 17 September 2015
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</span>
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Figure 1 illustrates the principle.  This method would besides providing quantitative information of the peptide aptamers affinity towards the target, also provides information of its specificity.
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</p>
  
Do we think of flying cars using Shawyer's <span class="tooltipLink">EmDrive</span><span class="tooltip"><span class="tooltipHeader">EmDrive</span>electromagnetic thruster</span>, Global Robot Rights or
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<span class="intro">Since we have been working with <i>E. coli</i> BTH101</span>, a β-Galactosidase Assay would also be an option for examine the affinity of the peptide aptamer towards the target. The assay exploits β –Galactosidase’s ability to recognize the synthetic compound o-nitrophenyl-β-D-galactoside (ONPG). The enzyme cleaves ONPG to galactose and o-nitrophenol, which has a yellow color. The production of yellow color can be used to determine the concentration of the enzyme, since when ONPG is in excess to the enzyme, the production of o-nitrophenol is proportional to the concentration of β-Galactosidase. From the assay, the miller unit can be determined. The miller unit for a known interaction in the system, e.g. our control with the Leuzine Zipper, could be used as a stand that peptide aptamers-target interaction could be compared <span class="sourceReference">to</span>.
human civilization on distant planets? All of the above and all other thoughts of the future are mental constructions inferred from the past. One might argue that the material past and the material future are exactly the same just wrapped in different semantic paradigms. It is actually impossible not be in the future, but the future is constituted by the immediate past, and the now is impossible to grasp. So is the past actually the future?</p>
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<span class="tooltip">
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  <span class="tooltipHeader">Reference:</span>
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    Open WetWare contributors, "Beta-Galactosidase Assay (A better Miller)," <i>Open WetWare</i>27 August 2012, 23:20 UTC.  <a target="blank_" href="http://openwetware.org/wiki/Beta-Galactosidase_Assay_(A_better_Miller)">http://openwetware.org/wiki/Beta-Galactosidase_Assay_(A_better_Miller)
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Accessed 17 September 2015.
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</p>
  
 
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<p>
You are always welcome to contact us by e-mail igemsdu2015 at gmail dot com, or visit one of our sites on the social medias.</p>
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<span class="intro">The peptide aptamer’s specificity </span> could be examined with protein microarray. Many different proteins would be immoblized to the microarry chip, the peptid aptamer taged with flourecent dye though anti-FLAG, would be added. Thus the fewer proteins towards the peptid aptamer can bind, the higher specificity.
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Latest revision as of 16:25, 4 October 2015

"The future belongs to those who believe in the beauty of their dreams." - Eleanor Roosevelt

Future Laboratory Work

After a screen with the nucleotide library and finding a peptide aptamer against a target protein the next step would be to examine the features of it.

The peptide aptamers affinity and specificity towards its target are two important parameters to know, especially if it is meant for use in diagnostics and treatment.

The affinity is especially important if we should get more than one positive colony in a screen. Several ways exist, to where we can test this, here amongs competitive Enzyme-Linked Immunosorbent Assay (ELISA). The ELISA wells would be coated with the target protein, to this we will add the peptide aptamer, giving them time to bind. After binding we will wash out any unbound peptide aptamers. A detection molecule that binds to the peptide aptamer though anti-FLAG will be added and later excess detection molecule will be washed out. An enzyme that converts the detections molecule to a color will be added. The color change or the amount of light emitted is proportional to the level of peptide aptamer bound to target. Reference: Technical Guide for ELISA. Available from KPL, Kirkegaard & Perry Laboratories. (Link) Accessed 17 September 2015 Thus the amount of peptide aptamer bound to target protein, provides a measurement for the affinity. The ELISA is competitive, thus the amount of target is realavlively small compared to that of peptide aptamers, this means the peptide aptamers will have to “compete” for binding the target protein.

Figure 1: Illustrates the detector with the Sensor chip, as the aqueous solution containing the target proteins flows by.

Surface Plasmon Resonance (SPR) is a technology designed by Biacore and could be used to investigate the affinity and the specificity of the peptide aptamer. Central for the SPR method is the movable sensor chip. This chip monitors the interaction between the peptide aptamer and its target. The operation of the instrument, data and the analysis of the data will be handled by a software. The sensor chip consist of a glass surface covered with a thin layer of gold, which forms a good basis to optimize the binding of a variety of molecules. To this chip the peptide aptamer would be bound. The target proteins will pass over the surface of the chip in a continuous, pulse-free and controlled flow, maintaining constant target protein concentration at the sensor chip surface. SPR measures changes in refractive index, thus senses changes in mass in the aqueous layer close to the sensor chip. Reference: An Introduction to BiaCore's SPR Technology. Available from BiaCore.com (Link) Accessed 17 September 2015 Figure 1 illustrates the principle. This method would besides providing quantitative information of the peptide aptamers affinity towards the target, also provides information of its specificity.

Since we have been working with E. coli BTH101, a β-Galactosidase Assay would also be an option for examine the affinity of the peptide aptamer towards the target. The assay exploits β –Galactosidase’s ability to recognize the synthetic compound o-nitrophenyl-β-D-galactoside (ONPG). The enzyme cleaves ONPG to galactose and o-nitrophenol, which has a yellow color. The production of yellow color can be used to determine the concentration of the enzyme, since when ONPG is in excess to the enzyme, the production of o-nitrophenol is proportional to the concentration of β-Galactosidase. From the assay, the miller unit can be determined. The miller unit for a known interaction in the system, e.g. our control with the Leuzine Zipper, could be used as a stand that peptide aptamers-target interaction could be compared to. Reference: Open WetWare contributors, "Beta-Galactosidase Assay (A better Miller)," Open WetWare27 August 2012, 23:20 UTC. http://openwetware.org/wiki/Beta-Galactosidase_Assay_(A_better_Miller) Accessed 17 September 2015.

The peptide aptamer’s specificity could be examined with protein microarray. Many different proteins would be immoblized to the microarry chip, the peptid aptamer taged with flourecent dye though anti-FLAG, would be added. Thus the fewer proteins towards the peptid aptamer can bind, the higher specificity.