Difference between revisions of "Team:NCTU Formosa/Results"

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<h1>Cell Staining Experiment</h1>
 
<h1>Cell Staining Experiment</h1>
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<p>After creating the parts of scFv and transforming them into our E.Cotector, we were going to prove that our detectors have successfully displayed scFv of anti-EGFR, anti-VEGF and anti-HER2 respectively. To prove this, we have decided to undergo the cell staining experiment by using our E.Cotector to detect the specific markers in the cell lines. </p>
+
<p>After creating the parts of scFv and transforming them into our E.Cotector, we were going to prove that our E.Cotectors have successfully displayed scFv of anti-EGFR, anti-VEGF and anti-HER2 respectively. We have decided to undergo cell staining experiment by using our E.Cotector to detect the specific markers in the cell lines. </p>
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<p>First of all, the main materials that we needed are fluorescent <I>E. coli</I> with various kinds of scFv, fluorescent <I>E. coli</I> without scFv and the cancer cell line – SKOV-3 that expressed or displayed the specific antigens for staining used. SKOV-3 is a kind of epithelial cell that expressed markers such as EGFR, VEGF and HER2.</p>
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<p>First of all, we need fluorescent <I>E. coli</I> with various kinds of scFv as experimental groups, fluorescent <I>E. coli</I> as control groups, and the cancer cell line – SKOV-3 that expressed the specific antigens such as EGFR, VEGF and HER2 for staining used.</p>
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<p>Each type of E. Cotector has been co-transformed with two different fluorescent colors ---RFP and GFP. Because of that, the negative control of this experiment are the red and green fluorescent <I>E. coli</I> without scFv expression. </p>
+
<p>In the cell staining experiment, the first thing is to fix the SKOV-3 on the 24 well plates. Next, we injected <I>E. coli</I> which diluted to OD600=1.6 into each well.
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<p>After injecting <I>E. coli</I> into the wells, we had to shake the plate in darkness for 45minutes. After staining for 45 minutes, we will wash away the unbind <I>E. coli</I> with PBS solution for a few times before observing the staining result under fluorescent microscope. Our result are as below:</p>
+
After injecting, we had to shake the plate in darkness for 45minutes. After staining for 45 minutes, we will wash away the unbind <I>E. coli</I> with PBS solution for a few times before observing the staining result under fluorescent microscope. Our result are as below:</p>
  
 
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<img src="https://static.igem.org/mediawiki/2015/d/de/NCTU_Formosa_Rusult1.png"height="200px"><br><br>
 
<img src="https://static.igem.org/mediawiki/2015/d/de/NCTU_Formosa_Rusult1.png"height="200px"><br><br>
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Figure 1. There is no <I>E. coli</I> stick on the cell’s surface.<br><br>
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<img src="https://static.igem.org/mediawiki/2015/7/73/NCTU_Formosa_Rusult2.png" height="200px"><br><br>
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In figure 2.3.4 the red fluorescent E.Cotectors bind to the corresponding makers. However, in figure 1 none of the red fluorescent <I>E. coli</I> bind on to the SKOV-3  <br><br>
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Figure 2. E. Cotector with anti-HER2 successfully stick on the cell’s surfaces as the scFv of anti-HER2 bind           to the HER2.<br><br>
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<img src="https://static.igem.org/mediawiki/2015/7/73/NCTU_Formosa_Rusult2.png" height="200px"><br><br>In figure 6.7.8 the green fluorescent E.Cotectors bind to the corresponding makers. However, in figure 5 none of the green fluorescent <I>E. coli</I> bind on to the SKOV-3 <br><br>
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<img src="https://static.igem.org/mediawiki/2015/0/0e/NCTU_Formosa_Rusult3.png"height="200px"><br><br>
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Figure 3. E. Cotector with anti-EGFR successfully stick on the cell’s surfaces as the scFv of anti-EGFR bind          to the EGFR.  <br><br>
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<img src="https://static.igem.org/mediawiki/2015/5/54/NCTU_Formosa_Rusult4.png" height="200px"><br><br>
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Figure 4.  E. Cotector with anti-VEGF successfully stick on the cell’s surfaces as the scFv of anti-VEGF bind          to the VEGF. 
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<h1>GBP Experiment</h1>
 
<h1>GBP Experiment</h1>
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<p>To verify that gold-binding polypeptide can bind on gold surface more efficiently, we created the genetic sequence, P<sub>induced</sub>+ rbs + FadL-GBP + RBS + GFP + ter, as the test group. Via the transmembrane protein FadL, the three-repeated gold-binding polypeptide can be displayed outside of the <i>E.coli</i> to recognize the gold surface. We intended to make the observation of the result more directly by producing green fluorescence at the same time. To make the comparison of the binding efficiency, we selected the genetic sequence, Pcons + rbs +GFP +ter, as the negative control group. Because there was no existence of fully-functioned phenomenon, there was no positive control in our experiment.</p>
+
<p>To prove the function of our extra library GBP, we use IPTG to induce promoter to express GBP in green fluorescent <i>E.coli</i> as control groups.
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<p>After culturing, we substituted the PBS buffer for the LB broth, and adjusted both the solution of test group and the control group to the same concentration. Then, we put the gold chip into the eppendofs which contain the prepared solutions and kept the eppendofs in the incubator at 25 degree celcius. After taking out the eppendofs, we washed each gold chips via the rotary machine for the purpose of maintaining the same washing condition. Subsequently, we classified the control groups and the test group, and settled them on the slides, just shown below. Then, we observed the results by the fluorescent microscopy. </p>
+
</p>
 +
<p>After culturing <i>E.coli</i>, we substituted PBS buffer for the LB broth, and adjusted both solution of experimental groups and the control groups to the same concentration. Then, we put the gold chip into the Eppendorf which contain the prepared solutions and kept the Eppendorf in the incubator at 25 degree Celsius. After taking out the Eppendorf, we washed each gold chips via the rotary machine for the purpose of maintaining the same washing condition. Subsequently, we classified the control groups and the experimental groups, and settled them on the slides, just shown below. Then, we observed the results by the fluorescent microscopy. </p>
 
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<img src="https://static.igem.org/mediawiki/2015/d/d8/Nctu_formosa_GBP_result_1.jpg" height="400px"><br><br>
 
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<p>According to the results shown below, we find out that there is more <i>E.coli</i> with gold-binding polypeptide being observed on the gold chip, which indicates that the <i>E.coli</i> with gold-binding polypeptide can attach on gold chip more effectively and efficiently than others without gold-binding polypeptide.</p>
+
<p>According to the results shown below, we find out that there is more <i>E.coli</i> with gold-binding polypeptide being observed on the gold chip, which indicates that the <i>E.coli</i> with gold-binding polypeptide can attach on gold chip more effectively and efficiently than the green fluorescent <i>E.coli</i>.</p>
 
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<a href="https://static.igem.org/mediawiki/2015/1/1b/Nctu_formosa_gbp_result_3a1.png"><img src="https://static.igem.org/mediawiki/2015/1/1b/Nctu_formosa_gbp_result_3a1.png" height="345px"></a>
 
<a href="https://static.igem.org/mediawiki/2015/1/1b/Nctu_formosa_gbp_result_3a1.png"><img src="https://static.igem.org/mediawiki/2015/1/1b/Nctu_formosa_gbp_result_3a1.png" height="345px"></a>
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<p>In magnification of 400,  figure (a) displays the consequence of the control group with the genetic sequence, P<sub>cons</sub>+ RBS + GFP + Ter while figure (b) shows the results of the test group, which contains the genetic sequence of P<sub>induced</sub>+ RBS + FadL-GBP + RBS + GFP + Ter. According to the pictures shown above, we can see that there is more <i>E.coli</i> with gold-binding polypeptide being observed on the gold chip. It indicates that the <i>E.coli</i> with gold-binding polypeptide can attach on gold chip more effectively and efficiently than others without gold-binding polypeptide.</p>
+
<p>In magnification of 400,  figure (a) displays the consequence of the control groups with the genetic sequence, P<sub>cons</sub>+ RBS + GFP + Ter while figure (b) shows the results of the experimental groups, which contains the genetic sequence of P<sub>induced</sub>+ RBS + FadL-GBP + RBS + GFP + Ter. According to the pictures shown above, we can see that there is more <i>E.coli</i> with gold-binding polypeptide being observed on the gold chip. It indicates that the <i>E.coli</i> with gold-binding polypeptide can attach on gold chip more effectively and efficiently than others without gold-binding polypeptide.</p>
 
</div>
 
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Revision as of 16:38, 18 September 2015

Results

Cell Staining Experiment

After creating the parts of scFv and transforming them into our E.Cotector, we were going to prove that our E.Cotectors have successfully displayed scFv of anti-EGFR, anti-VEGF and anti-HER2 respectively. We have decided to undergo cell staining experiment by using our E.Cotector to detect the specific markers in the cell lines.

First of all, we need fluorescent E. coli with various kinds of scFv as experimental groups, fluorescent E. coli as control groups, and the cancer cell line – SKOV-3 that expressed the specific antigens such as EGFR, VEGF and HER2 for staining used.

In the cell staining experiment, the first thing is to fix the SKOV-3 on the 24 well plates. Next, we injected E. coli which diluted to OD600=1.6 into each well. After injecting, we had to shake the plate in darkness for 45minutes. After staining for 45 minutes, we will wash away the unbind E. coli with PBS solution for a few times before observing the staining result under fluorescent microscope. Our result are as below:



In figure 2.3.4 the red fluorescent E.Cotectors bind to the corresponding makers. However, in figure 1 none of the red fluorescent E. coli bind on to the SKOV-3



In figure 6.7.8 the green fluorescent E.Cotectors bind to the corresponding makers. However, in figure 5 none of the green fluorescent E. coli bind on to the SKOV-3

GBP Experiment

To prove the function of our extra library GBP, we use IPTG to induce promoter to express GBP in green fluorescent E.coli as control groups.

After culturing E.coli, we substituted PBS buffer for the LB broth, and adjusted both solution of experimental groups and the control groups to the same concentration. Then, we put the gold chip into the Eppendorf which contain the prepared solutions and kept the Eppendorf in the incubator at 25 degree Celsius. After taking out the Eppendorf, we washed each gold chips via the rotary machine for the purpose of maintaining the same washing condition. Subsequently, we classified the control groups and the experimental groups, and settled them on the slides, just shown below. Then, we observed the results by the fluorescent microscopy.



Figure 1.



Figure 2.

According to the results shown below, we find out that there is more E.coli with gold-binding polypeptide being observed on the gold chip, which indicates that the E.coli with gold-binding polypeptide can attach on gold chip more effectively and efficiently than the green fluorescent E.coli.



In magnification of 400, figure (a) displays the consequence of the control groups with the genetic sequence, Pcons+ RBS + GFP + Ter while figure (b) shows the results of the experimental groups, which contains the genetic sequence of Pinduced+ RBS + FadL-GBP + RBS + GFP + Ter. According to the pictures shown above, we can see that there is more E.coli with gold-binding polypeptide being observed on the gold chip. It indicates that the E.coli with gold-binding polypeptide can attach on gold chip more effectively and efficiently than others without gold-binding polypeptide.



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