Difference between revisions of "Team:CGU Taiwan/Notebook"

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<table class="protocol-table" style="margin:auto">
 
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<tr><td><a>Description</a></td><td><a>Yeast With IL-8 Receptor</a></td><td><a>Toehold Switch As RNA Senor</a></td><td><a>Prototype Design</a></td></tr>
 
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<div class="single-blog blog-details two-column" id="Protocols">
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<h2 class="post-title bold">GPCR</h2>
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<div id="accordion-container">
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<div class="panel-heading">
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<h4 class="panel-title">
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<a data-toggle="collapse" data-parent="#accordion" href="#collapse1">2015.7.1</a>
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</div>
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<div id="collapse1" class="panel-collapse collapse ">
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<div class="panel-body">
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Operator: Wan-Yun, Jin-Ting, Jin-Ye<br>
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Goal:
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<br>
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&nbsp;&nbsp;1. Extraction of gDNA of Far1∆::KANMX strain<br>
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&nbsp;&nbsp;2. Detection the concentration of fast extracted gDNA of ∆Far1 strain<br>
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&nbsp;&nbsp;3. PCR gDNA of Far1∆ ::KANMX<br>
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&nbsp;&nbsp;4. Electrophoresis to check PCR product<br>
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<br>
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Experiment steps:<br>
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−
< Extraction of gDNA of Far1∆::KANMX strain><br>
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−
Consult the protocol<protocol of fast extraction of gDNA of yeast><br>
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<Detection the concentration of fast extracted gDNA>
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.home-box2 a {
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<table class="protocol-table">
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<tr><th></th><th></th><th></th><th></th></tr>
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text-align:center;
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<tr><td>Strains </td><td>260/280 </td><td>260/230 </td><td>C(ng/μl) </td></tr>
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}
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<tr><td>Far1∆::KANMX strain </td><td>1.62 </td><td>0.97 </td><td>44.7 </td></tr>
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</table>
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<br>
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<PCR gDNA of Far1∆::KANMX ><br>
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1. Design of primers<br>
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<table class="protocol-table">
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<thead>
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<tr><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th></tr>
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</thead>
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<tr><td> Name</td><td>Pur. </td><td>Seq.(5’-3’) </td><td>Size (mer.) </td><td> MW (g/mol)</td><td>Tm (℃)</td><td>GC (%)</td><td>Nmole</td><td>μl for 100μM </td></tr>
+
−
<tr><td>dFAR1 F’</td><td>Desalt </td><td>ggTTTTgTTAggCgggCAAg </td><td>20 </td><td>6244.1 </td><td>53.8 </td><td>55</td><td>21.25</td><td>212.50</td></tr>
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<tr><td>dFAR1 R’</td><td>Desalt </td><td>CATTAACTgCTATTTACgACgC </td><td>22 </td><td>6669.4 </td><td>51.1 </td><td>41</td><td>17.12</td><td>171.20</td></tr>
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</table>
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<br>
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2. PCR program
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<table class="protocol-table">
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<thead>
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<tr><th></th><th></th><th></th></tr>
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</thead>
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<tr><td>Step </td><td>Temperature </td><td>Time</td></tr>
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<tr><td>Step1 </td><td>95℃ </td><td>5min</td></tr>
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<tr><td>Step2 </td><td>95℃ </td><td>30s</td></tr>
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<tr><td>Step3 </td><td>52℃ </td><td>30s</td></tr>
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<tr><td>Step4→step2 for 30 cycle </td><td>72℃ </td><td>2min </td></tr>
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<tr><td>Step5 </td><td>72℃ </td><td>5min </td></tr>
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<tr><td>Step6 (hold on) </td><td>10℃ </td><td>1hr </td></tr>
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</table>
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<br>
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−
3.PCR reagent
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<table class="protocol-table">
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<thead>
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−
<tr><th></th><th></th></tr>
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</thead>
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<tr><td>10x Dream Taq buffer </td><td>2.5μl</td></tr>
+
−
<tr><td>2.5mM dNTP</td><td>0.5μl</td></tr>
+
−
<tr><td>10mM primer(F)</td><td>0.5μl</td></tr>
+
−
<tr><td>10mM primer(R)</td><td>0.5μl </td></tr>
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−
<tr><td>template (Far1∆::KANMX strain gDNA) </td><td>3.4μl</td></tr>
+
−
<tr><td>Taq polymerase</td><td>0.5μl</td></tr>
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−
<tr><td>ddH2O </td><td>17.1μl</td></tr>
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−
<tr><td>Total volume</td><td>25μl</td></tr>
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−
</table><br>
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<br>
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−
< Electrophoresis to check PCR product><br>
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−
Material: <br>
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−
&nbsp;&nbsp;DNA marker: 100bp ladder 8μl<br>
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−
&nbsp;&nbsp;DNA sample: 2μlPCR product + 1μl 6x loading buffer<br>
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Condition: <br>
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&nbsp;&nbsp;0.5xTBE buffer 100V<br>
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−
Time:<br>
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−
&nbsp;&nbsp;30min<br>
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Result:<br>
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−
<!--照片-->
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−
&nbsp;&nbsp;M：Marker；#1：Far1 ∆ for annealing at 52℃<br>
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−
&nbsp;&nbsp;There is no band appears in the gel electrophoresis.<br>
+
−
Conclusion:<br>
+
−
&nbsp;&nbsp;Due to the Figure 1 in result, we have to check the temperature of primers annealing and the design of primers to solve the problem. Next, we use gradient PCR to find out the exactly temperature of primer annealing and we add positive control as well.
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<div class="panel-heading">
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<h4 class="panel-title">
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<a data-toggle="collapse" data-parent="#accordion" href="#collapse2">2015.7.2</a>
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</h4>
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</div>
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<div id="collapse2" class="panel-collapse collapse ">
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<div class="panel-body">
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Operator: Wan-Yun, Jin-Ting, Jin-Ye<br>
+
−
Goal: <br>
+
−
&nbsp;&nbsp;1. Extraction of gDNA of FAR1∆::KANMX strain <br>
+
−
&nbsp;&nbsp;2. Detection the concentration of fast extracted gDNA of FAR1∆::KANMX strain<br>
+
−
&nbsp;&nbsp;3. First round of PCR <br>
+
−
&nbsp;&nbsp;4. Electrophoresis to check first round-PCR product<br>
+
−
&nbsp;&nbsp;5. Second round of PCR <br>
+
−
<br>
+
−
Experiment steps:<br>
+
−
< Extraction of gDNA of FAR1∆::KANMX strain><br>
+
−
Consult the protocol <protocol of fast extraction of gDNA of yeast><br>
+
  
 +
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<Detection the concentration of fast extracted gDNA>
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<table class="protocol-table">
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</style>
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<thead>
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−
<tr><th></th><th></th><th></th><th></th></tr>
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−
</thead>
+
−
<tr><td>Strains </td><td>260/280 </td><td>260/230 </td><td>C(ng/μl) </td></tr>
+
−
<tr><td>Far1∆::KANMX </td><td>1.72 </td><td>0.78 </td><td>42.7 </td></tr>
+
−
<tr><td>Positive control </td><td>1.63 </td><td>0.76 </td><td>37.1 </td></tr>
+
−
</table>
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−
+
−
<First round of PCR ><br>
+
−
1. Information of primers<br>
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<table class="protocol-table">
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−
<thead>
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−
<tr><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th></tr>
+
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</thead>
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−
<tr><td> Name</td><td>Pur. </td><td>Seq.(5’-3’) </td><td>Size (mer.) </td><td> MW (g/mol)</td><td>Tm (℃)</td><td>GC (%)</td><td>Nmole</td><td>μl for 100μM </td></tr>
+
−
<tr><td>dFAR1 F’</td><td>Desalt </td><td>ggTTTTgTTAggCgggCAAg </td><td>20 </td><td>6244.1 </td><td>53.8 </td><td>55</td><td>21.25</td><td>212.50</td></tr>
+
−
<tr><td>dFAR1 R’</td><td>Desalt </td><td>CATTAACTgCTATTTACgACgC </td><td>22 </td><td>6669.4 </td><td>51.1 </td><td>41</td><td>17.12</td><td>171.20</td></tr>
+
−
</table>
+
−
<br>
+
−
2.PCR program<br>
+
−
<table class="protocol-table">
+
−
<thead>
+
−
<tr><th></th><th></th><th></th></tr>
+
−
</thead>
+
−
<tr><td>Step </td><td>Temperature </td><td>Time</td></tr>
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−
<tr><td>Step1 </td><td>95℃ </td><td>5min</td></tr>
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−
<tr><td>Step2 </td><td>95℃ </td><td>30s</td></tr>
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−
<tr><td>Step3 </td><td>Gradient42℃-46℃-50℃ </td><td>30s</td></tr>
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<tr><td>Step4→step2 for 30 cycle </td><td>72℃ </td><td>2min </td></tr>
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−
<tr><td>Step5 </td><td>72℃ </td><td>5min </td></tr>
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−
<tr><td>Step6 (hold on) </td><td>10℃ </td><td>1hr </td></tr>
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−
</table>
+
−
<br>
+
−
3.PCR reagent<br>
+
−
<table class="protocol-table">
+
−
<thead>
+
−
<tr><th></th><th></th></tr>
+
−
</thead>
+
−
<tr><td>10x Dream Taq buffer </td><td>2.5μl</td></tr>
+
−
<tr><td>2.5mM dNTP</td><td>0.5μl</td></tr>
+
−
<tr><td>10mM primer(F)</td><td>0.5μl</td></tr>
+
−
<tr><td>10mM primer(R)</td><td>0.5μl </td></tr>
+
−
<tr><td>template (Far1∆::KANMX strain gDNA) </td><td>3.4μl</td></tr>
+
−
<tr><td>Taq polymerase</td><td>0.5μl</td></tr>
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<tr><td>ddH2O </td><td>17.1μl</td></tr>
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<tr><td>Total volume</td><td>25μl</td></tr>
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</table><br>
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<br>
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< Electrophoresis to check first round-PCR product (25μl ul)><br>
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Material:<br>
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&nbsp;&nbsp;DNA marker: 100bp ladder 8μl<br>
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&nbsp;&nbsp;DNA sample: 2μl PCR product + 1μl 6x loading buffer<br>
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Condition: <br>
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&nbsp;&nbsp;0.5xTBE buffer 100V <br>
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Time: <br>
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&nbsp;&nbsp;30min<br>
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Result:<br>
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<!--照片-->
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M: Marker; #1: FAR1∆ annealing at 42 ℃; #2: FAR1∆ annealing at 46 ℃;<br>
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#3: FAR1∆ annealing at 50 ℃; #4: FAR1∆ annealing at 52 ℃(positive control)
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<div class="panel panel-default">
 
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<div class="panel-heading">
 
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<h4 class="panel-title">
 
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<a data-toggle="collapse" data-parent="#accordion" href="#collapse1">2015.7.1</a>
 
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</h4>
 
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</div>
 
−
<div id="collapse1" class="panel-collapse collapse ">
 
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<div class="panel-body">
 
−
Operator: Wan yun, Jinting , Jinye
 
−
Goal:
 
−
1. Extraction of gDNA of Far1∆::KANMX strain
 
−
    2. Detection the concentration of fast extracted gDNA of ∆Far1 strain
 
−
3. PCR gDNA of Far1∆ ::KANMX
 
−
4. Electrophoresis to check PCR product
 
−
Experiment steps:
 
−
< Extraction of gDNA of Far1∆::KANMX strain>
 
−
Consult the protocol<protocol of fast extraction of gDNA of yeast>
 
  
−
<Detection the concentration fo fast extracted gDNA>
 
−
<table class="protocol-table">
 
−
<thead>
 
−
<tr><th></th><th></th></tr>
 
−
</thead>
 
−
<tr><td>Strains </td><td>260/280 </td><td>260/230 </td><td>C(ng/μl) </td></tr>
 
−
<tr><td>Far1∆::KANMX strain </td><td>1.62 </td><td>0.97 </td><td>44.7 </td></tr>
 
−
</table>
 
−
 
−
<PCR gDNA of Far1∆::KANMX >
 
−
1. Design of primers
 
−
<table class="protocol-table">
 
−
<thead>
 
−
<tr><th></th><th></th></tr>
 
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</thead>
 
−
<tr><td> </td><td> </td><td> </td><td> </td><td> </td><td>Tm (℃)</td><td>GC (%)</td><td>Nmole</td><td>μl for 100μM </td></tr>
 
−
<tr><td> </td><td> </td><td> </td><td> </td><td> </td><td> </td><td>55</td><td>21.25</td><td>212.50</td></tr>
 
−
<tr><td> </td><td> </td><td> </td><td> </td><td> </td><td> </td><td>41</td><td>17.12</td><td>171.20</td></tr>
 
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</table>
 
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<table class="protocol-table">
 
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<thead>
 
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<tr><th></th><th></th></tr>
 
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<div class="panel panel-default">
 
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<div class="panel-heading">
 
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<h4 class="panel-title">
 
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<a data-toggle="collapse" data-parent="#accordion" href="#collapse1">2015.7.1</a>
 
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</h4>
 
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</div>
 
−
<div id="collapse1" class="panel-collapse collapse ">
 
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<div class="panel-body">
 
−
Operator: Wan yun, Jinting , Jinye
 
−
Goal:
 
−
1. Extraction of gDNA of Far1∆::KANMX strain
 
−
    2. Detection the concentration of fast extracted gDNA of ∆Far1 strain
 
−
3. PCR gDNA of Far1∆ ::KANMX
 
−
4. Electrophoresis to check PCR product
 
−
Experiment steps:
 
−
< Extraction of gDNA of Far1∆::KANMX strain>
 
−
Consult the protocol<protocol of fast extraction of gDNA of yeast>
 
  
−
<Detection the concentration fo fast extracted gDNA>
+
<body>
−
<table class="protocol-table">
+
−
<thead>
+
−
<tr><th></th><th></th></tr>
+
−
</thead>
+
−
<tr><td>Strains </td><td>260/280 </td><td>260/230 </td><td>C(ng/μl) </td></tr>
+
−
<tr><td>Far1∆::KANMX strain </td><td>1.62 </td><td>0.97 </td><td>44.7 </td></tr>
+
−
</table>
+
−
+
−
<PCR gDNA of Far1∆::KANMX >
+
−
1. Design of primers
+
−
<table class="protocol-table">
+
−
<thead>
+
−
<tr><th></th><th></th></tr>
+
−
</thead>
+
−
<tr><td> </td><td> </td><td> </td><td> </td><td> </td><td>Tm (℃)</td><td>GC (%)</td><td>Nmole</td><td>μl for 100μM </td></tr>
+
−
<tr><td> </td><td> </td><td> </td><td> </td><td> </td><td> </td><td>55</td><td>21.25</td><td>212.50</td></tr>
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<tr><td> </td><td> </td><td> </td><td> </td><td> </td><td> </td><td>41</td><td>17.12</td><td>171.20</td></tr>
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Operator: Wan yun, Jinting , Jinye
+
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Goal:
+
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1. Extraction of gDNA of Far1∆::KANMX strain
+
−
    2. Detection the concentration of fast extracted gDNA of ∆Far1 strain
+
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3. PCR gDNA of Far1∆ ::KANMX
+
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4. Electrophoresis to check PCR product
+
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Experiment steps:
+
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< Extraction of gDNA of Far1∆::KANMX strain>
+
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Consult the protocol<protocol of fast extraction of gDNA of yeast>
+
  
−
<Detection the concentration fo fast extracted gDNA>
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    <section id="recent-projects" class="recent-projects">
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<tr><td>Strains </td><td>260/280 </td><td>260/230 </td><td>C(ng/μl) </td></tr>
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                <h1 class="title text-center wow fadeInDown" data-wow-duration="500ms" data-wow-delay="300ms">Welcome to CGU_Taiwan's Wiki page</h1>
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<tr><td>Far1∆::KANMX strain </td><td>1.62 </td><td>0.97 </td><td>44.7 </td></tr>
+
                <h2 class="text-center padding-bottom wow fadeInDown" data-wow-duration="400ms" data-wow-delay="400ms">Oral cancer is a common cancer worldwide. One of the major problems of oral cancer is that early malignancy could only be detected by clinical oral examination from health care professionals, and there are no clinical tests at the molecular level. Recently, there are several Proteins and mRNAs reported as promising potential non-invasive biomarkers in saliva for oral cancer. Our team wants to establish simple, objective and quantifiable non-invasive detection system for those biomarkers.
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<br>Our system includes two aims :
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+
<br>1) engineered yeasts that express a reporter activated by IL-8, which is one of the most statistically significant protein biomarkers in oral cancer saliva samples.
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<PCR gDNA of Far1∆::KANMX >
+
<br>2) Synthetic toehold switch gene regulators as RNA sensors to detect specific mRNA biomarkers. These efforts will provide the groundwork for new molecular diagnoses in oral cancer.</h2>
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1. Design of primers
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<tr><td> </td><td> </td><td> </td><td> </td><td> </td><td> </td><td>55</td><td>21.25</td><td>212.50</td></tr>
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<tr><td> </td><td> </td><td> </td><td> </td><td> </td><td> </td><td>41</td><td>17.12</td><td>171.20</td></tr>
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<a data-toggle="collapse" data-parent="#accordion" href="#collapse1">2015.7.1</a>
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Operator: Wan yun, Jinting , Jinye
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<div class="home-slider2 portfolio-wrapper">
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<div class="home-slider3 portfolio-single">
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1. Extraction of gDNA of Far1∆::KANMX strain
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<div class="home-box1">
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    2. Detection the concentration of fast extracted gDNA of ∆Far1 strain
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<img src="https://static.igem.org/mediawiki/2015/2/2f/CGU-home-logo.jpg" >
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3. PCR gDNA of Far1∆ ::KANMX
+
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4. Electrophoresis to check PCR product
+
<div class="home-box2">
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Experiment steps:
+
<img src="https://static.igem.org/mediawiki/2015/5/51/CGU-home-HP-258x266.jpeg">
−
< Extraction of gDNA of Far1∆::KANMX strain>
+
</div>
−
Consult the protocol<protocol of fast extraction of gDNA of yeast>
+
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 +
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<li>&nbsp;&nbsp;&nbsp;&nbsp;Project</li>
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<li>&nbsp;&nbsp;&nbsp;&nbsp;Experiment</li>
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<li>Parts</li>
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<li>Human&nbsp;Practice</li>
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<div class="home-menu1">  
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<li>&nbsp;&nbsp;&nbsp;&nbsp;Collaboration</li>
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<li>&nbsp;&nbsp;&nbsp;&nbsp;About Us</li>
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<li>Attribution</li>
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<li>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Note</li>
 +
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    <!--/#recent-projects-->
  
−
<Detection the concentration fo fast extracted gDNA>
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    <section id="clients">
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<tr><td>Strains </td><td>260/280 </td><td>260/230 </td><td>C(ng/μl) </td></tr>
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                        <p><img src="images/home/clients.png" class="img-responsive" alt=""></p>
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<tr><td>Far1∆::KANMX strain </td><td>1.62 </td><td>0.97 </td><td>44.7 </td></tr>
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                        <h1 class="title">~~~~~~~~~~~</h1>
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                        <p>~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ <br>~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ </p>
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                    </div>
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<PCR gDNA of Far1∆::KANMX >
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                    <div class="clients-logo wow fadeIn" data-wow-duration="1000ms" data-wow-delay="600ms">
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1. Design of primers
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<tr><td> </td><td> </td><td> </td><td> </td><td> </td><td> </td><td>55</td><td>21.25</td><td>212.50</td></tr>
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<tr><td> </td><td> </td><td> </td><td> </td><td> </td><td> </td><td>41</td><td>17.12</td><td>171.20</td></tr>
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Operator: Wan yun, Jinting , Jinye
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Goal:
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1. Extraction of gDNA of Far1∆::KANMX strain
+
−
    2. Detection the concentration of fast extracted gDNA of ∆Far1 strain
+
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3. PCR gDNA of Far1∆ ::KANMX
+
−
4. Electrophoresis to check PCR product
+
−
Experiment steps:
+
−
< Extraction of gDNA of Far1∆::KANMX strain>
+
−
Consult the protocol<protocol of fast extraction of gDNA of yeast>
+
  
−
<Detection the concentration fo fast extracted gDNA>
 
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<tr><td>Strains </td><td>260/280 </td><td>260/230 </td><td>C(ng/μl) </td></tr>
 
−
<tr><td>Far1∆::KANMX strain </td><td>1.62 </td><td>0.97 </td><td>44.7 </td></tr>
 
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<PCR gDNA of Far1∆::KANMX >
 
−
1. Design of primers
 
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<tr><td> </td><td> </td><td> </td><td> </td><td> </td><td> </td><td>55</td><td>21.25</td><td>212.50</td></tr>
 
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<tr><td> </td><td> </td><td> </td><td> </td><td> </td><td> </td><td>41</td><td>17.12</td><td>171.20</td></tr>
 
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<div id="collapse1" class="panel-collapse collapse ">
 
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Operator: Wan yun, Jinting , Jinye
 
−
Goal:
 
−
1. Extraction of gDNA of Far1∆::KANMX strain
 
−
    2. Detection the concentration of fast extracted gDNA of ∆Far1 strain
 
−
3. PCR gDNA of Far1∆ ::KANMX
 
−
4. Electrophoresis to check PCR product
 
−
Experiment steps:
 
−
< Extraction of gDNA of Far1∆::KANMX strain>
 
−
Consult the protocol<protocol of fast extraction of gDNA of yeast>
 
  
−
<Detection the concentration fo fast extracted gDNA>
+
   
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<table class="protocol-table">
+
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+
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</thead>
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−
<tr><td>Strains </td><td>260/280 </td><td>260/230 </td><td>C(ng/μl) </td></tr>
+
−
<tr><td>Far1∆::KANMX strain </td><td>1.62 </td><td>0.97 </td><td>44.7 </td></tr>
+
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<PCR gDNA of Far1∆::KANMX >
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1. Design of primers
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Operator: Wan yun, Jinting , Jinye
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1. Extraction of gDNA of Far1∆::KANMX strain
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    2. Detection the concentration of fast extracted gDNA of ∆Far1 strain
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3. PCR gDNA of Far1∆ ::KANMX
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4. Electrophoresis to check PCR product
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< Extraction of gDNA of Far1∆::KANMX strain>
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<tr><td>Far1∆::KANMX strain </td><td>1.62 </td><td>0.97 </td><td>44.7 </td></tr>
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Home | CGU_Taiwan

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Oral cancer is a common cancer worldwide. One of the major problems of oral cancer is that early malignancy could only be detected by clinical oral examination from health care professionals, and there are no clinical tests at the molecular level. Recently, there are several Proteins and mRNAs reported as promising potential non-invasive biomarkers in saliva for oral cancer. Our team wants to establish simple, objective and quantifiable non-invasive detection system for those biomarkers.
Our system includes two aims :
1) engineered yeasts that express a reporter activated by IL-8, which is one of the most statistically significant protein biomarkers in oral cancer saliva samples.
2) Synthetic toehold switch gene regulators as RNA sensors to detect specific mRNA biomarkers. These efforts will provide the groundwork for new molecular diagnoses in oral cancer.

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