Team:NCTU Formosa/Results

Results

Customize Platform Experiment

We have the anti-EGFR, anti-VEGF, and anti-HER2 scFv to build the probe. The gel electrophoresis prove that length of insert gene is right. (Figure 1.)




Figure 1.

(A) This insert of scFv is anti-EGFR, as expected, that the base length was 1272 bp.

(B) This insert of scFv is anti-VGFR, as expected, that the base length was 1260 bp.

(C) This insert of scFv is anti-HER2, as expected, that the base length was 1061 bp.

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.(Figure 2.) Our result are as below:


Figure 2.

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

Figure 3.This insert, as expected, that the base length was 1272 bp

To prove the function of our extra library GBP, we use constitutive promoter to express GBP in green fluorescent E.coli as experimental groups and the 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 (Figure 4.) 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. (Figure 5.) Then, we observed the results by the fluorescent microscopy.



Figure 4.



Figure 5.

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.(Figure 6.)


Figure 6.

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 Pcons+ 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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