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

 
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<p> <i> "??." - <b>who??</b></i></p>
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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>
  
<h1 align="center"> Future work </h1>
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<h1 align="center"> Future Laboratory  Work </h1>
  
 
<p>
 
<p>
<span class="intro">After a screen with the nucleotide library </span> for a peptide aptamer against a target the next step would be to examine features of the peptide aptamer.
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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.
 
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<span class="intro">The affinity is especially important </span> to know, if we should get more than one positive in a screen. There are several ways to test this, here among competitive Enzyme-Linked Immunosorbent Assay (ELISA). The ELISA wells will be coated with the target protein, to this we will add the peptide aptamer and the unbound peptide aptamer will be washed out. A detection molecule that binds to the peptide aptamer though anti-FLAG will be added and 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="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>.
 
<span class="tooltip">
 
<span class="tooltip">
 
   <span class="tooltipHeader">Reference:</span>
 
   <span class="tooltipHeader">Reference:</span>
     Technical Guide for ELISA. <a target="blank_" href="http://www.kpl.com/docs/techdocs/KPL%20ELISA%20Technical%20Guide.pdf">(Link)
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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)
 
</a>
 
</a>
 
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Accessed 17 September 2015
 
</span>
 
</span>
  
(1).
 
  
 
Thus the amount of peptide aptamer bound to target protein, provides a measurement for the affinity.
 
Thus the amount of peptide aptamer bound to target protein, provides a measurement for the affinity.
That the ELISA is competitive, means that the amount of peptide aptamer are greater than target protein, thus the peptide aptamers will have to “compete” for binding the target protein.
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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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<a class="popupImg alignRight" style="width:230px" target="_blank" href="https://static.igem.org/mediawiki/2015/a/ad/Detector.png" title="Figure 1 illustrates the detector with the Sensor chip, as the aqueous solution containing the target proteins flows by (2).">
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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">
   <img src="https://static.igem.org/mediawiki/2015/a/ad/Detector.png" style="width:230px"/></a>
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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.
<div class="thumbcaption"><i>Figure 1:</i> SPR detector and sensor chip </div>
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<p>
<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 monitor the interaction between the peptide aptamer and the target. The operation of the instrument, the data and the analyze of the data will be handled by a software.
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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.
 
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 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.   
 
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 (2)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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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">
 +
  <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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</a>
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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.
 
</p>
 
</p>
  
 
<p>
 
<p>
<span class="intro">Since we have been working with E. coli 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. Instead of using X-gal as we have done in our previous experiments, we will in such assay use ONPG. The production of the 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 (3)
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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>.
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<span class="tooltip">
 +
  <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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</a>
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Accessed 17 September 2015.
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(1) Technical Guide for ELISA. Available at: http://www.kpl.com/docs/techdocs/KPL%20ELISA%20Technical%20Guide.pdf [Accessed: 17-09-15]
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</p>
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<p>
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(2) An Introduction to Biacore’s SPR Technology. Available at: http://www.rci.rutgers.edu/~longhu/Biacore/pdf_files/SPR_Technology_Brochure.pdf [Accessed: 17-09-15]   
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</p>
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<p>
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(3) Openwetware.org. Beta-Galactosidase Assay (A better Miller). Available at: http://openwetware.org/wiki/Beta-Galactosidase_Assay_(A_better_Miller) [Accessed: 17-09-15]     
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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.