Team:NCTU Formosa/Results

Results

Cell Staining Experiment

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.

First of all, the main materials that we needed are fluorescent E. coli with various kinds of scFv, fluorescent E. coli 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.

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 E. coli without scFv expression.

After injecting E. coli into the wells, 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:



Figure 1. There is no E. coli stick on the cell’s surface.



Figure 2. E. Cotector with anti-HER2 successfully stick on the cell’s surfaces as the scFv of anti-HER2 bind to the HER2.



Figure 3. E. Cotector with anti-EGFR successfully stick on the cell’s surfaces as the scFv of anti-EGFR bind to the EGFR.



Figure 4. E. Cotector with anti-VEGF successfully stick on the cell’s surfaces as the scFv of anti-VEGF bind to the VEGF.

GBP Experiment

To verify that gold-binding polypeptide can bind on gold surface more efficiently, we created the genetic sequence, Pinduced+ 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 E.coli 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.

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.



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 others without gold-binding polypeptide.



In magnification of 400, figure (a) displays the consequence of the control group with the genetic sequence, Pcons+ RBS + GFP + Ter while figure (b) shows the results of the test group, 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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