Difference between revisions of "Team:Hong Kong-CUHK/Results"

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<div id="section3" class="container-fluid">
 
<div id="section3" class="container-fluid">
 
   <h1>Characterization</h1>
 
   <h1>Characterization</h1>
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  <p>Overview
 
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<br>Fluorescent protein is an important marker for qualitative analysis in validation test of Toehold switch which detects the presence of trigger (viral) RNA.The presence of trigger RNA is reported by fold change of fluorescent protein expression compared with switch only by fluorescent assays and rely on the ability of fluorescent protein expression. More often, body fluid of different pH from vary organs extracted as sample may disturb the fluorescent level due to pH-dependent folding and conformation of proteins.
 
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<br>To determine the effective range and optimize the protein expression level for clinical diagnosis, we have demonstrated their pH stability under different pH condition.
 
  
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<br><br>Key Achievements
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We found that amajLime was described as “chromoprotein” in the main page. We later found that amajLime is a fluorescent protein instead. Therefore we scanned the emission and excitation spectrum of it and documented it in registry:
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<br>Characterised two fluorescent proteins for the registry to functionalize Toehold switch for clinical diagnosis.
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<img src="https://static.igem.org/mediawiki/parts/1/15/Ex-Em._amajlime.png" width="100%" height="auto">
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Proved the pH dependent stability of fluorescent proteins.</p>
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We then measured the fluorescent signal of mRFP and amajLime in buffer with different pH:
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<img src="
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https://static.igem.org/mediawiki/parts/5/56/Mrfp.PNG" width="100%" height="auto">
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<img src="https://static.igem.org/mediawiki/parts/4/44/Amaj.PNG" width="100%" height="auto">
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Purified mRFP and amajLime was diluted to 10µg/100µl (total 200µl), in triplicates, into different buffers which pH ranges from pH2 to pH12. Volume of mRFP: pH buffer=1:1.8, amajLime : pH buffer =1:4.3. The lines represent the trend of the median fluorescence for the measured pH values. mRFP and amajLime were excited by 584nm and 470nm respectively and emission was measured at 607nm and 515nm respectively. To allow repeatable experiment, we converted the relative fluorescent intensity to an absolute fluorophore concentration by plotting standard curve of the fluorophore using the interlab protocol. We used Rhodamine to measure mRFP; Fluorescein to measure amajLime. The results show that the optimum pH of both chromoprotein ranges from pH6 to pH8. Their fluorescent signal thus will probably not be interfered by the pH of our proposed sample: saliva or NP swab.
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</p>
 
</div>
 
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Revision as of 12:11, 22 October 2017




Results

Toehold Switch

This is overview about Toehold Switch.

Interlab

Please refer to the Interlab part.

Characterization

We found that amajLime was described as “chromoprotein” in the main page. We later found that amajLime is a fluorescent protein instead. Therefore we scanned the emission and excitation spectrum of it and documented it in registry: We then measured the fluorescent signal of mRFP and amajLime in buffer with different pH: Purified mRFP and amajLime was diluted to 10µg/100µl (total 200µl), in triplicates, into different buffers which pH ranges from pH2 to pH12. Volume of mRFP: pH buffer=1:1.8, amajLime : pH buffer =1:4.3. The lines represent the trend of the median fluorescence for the measured pH values. mRFP and amajLime were excited by 584nm and 470nm respectively and emission was measured at 607nm and 515nm respectively. To allow repeatable experiment, we converted the relative fluorescent intensity to an absolute fluorophore concentration by plotting standard curve of the fluorophore using the interlab protocol. We used Rhodamine to measure mRFP; Fluorescein to measure amajLime. The results show that the optimum pH of both chromoprotein ranges from pH6 to pH8. Their fluorescent signal thus will probably not be interfered by the pH of our proposed sample: saliva or NP swab.

Program

Program

     

Email: igemcuhk1617@gmail.com