Team:NCTU Formosa/Results

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To prove that our E.Coli have successfully displayed scFv of Bevacixumab and Cetuxicumab respectively and the high specificity of our E.Cotector, we designed cell staining experiment. The mainly material that we needed are fluorescent E.Coli with scFv and without scFv, the cancer cells that expressed or displayed the specific antigens for staining used.
Introduction of cell lines that we used:

1. MCF7 cell line:
MCF7 is a kind of epithelial cell of human breast cancer cells. There are overexpressed epithermal growth factor receptor (EGFR) on the cell surfaces.

2. MDA-MB-231 cell line:
MDA-MB-231 is also a kind of epithelial cell that can produce vascular epithermal growth factor (VEGF) that will trigger the abnormal growth of vascular, angiogenesis.
Before undergoing cell staining, we would cultivate MCF7 and MDA-MB-231 each in a 6-well plate. Besides, we would also cultivate our anti-EGFR E.Cotector, anti-VEGF E.Cotector and green fluorescent E.Coli as control in experiment for 18 hours to make sure that the concentration of E.Coli are good enough for undergoing cell staining. Before cell staining, we had to add triton into some wells to permeabilize the cell membrane of MDA-MB-231. The reason to do so is that VEGF is growth factor that is produced and released by cancer cells and not receptors that are displayed on those cell surfaces. After injecting E.Coli into the wells, we had shaken the plates in darkness for 1hour. After staining for 1 hour, we will wash away unbind E.Coli with PBS solution for a few times before observing the staining result under fluorescent microscope. In our hypothesis, if we abled to observe anti-VEGF E.Cotector penetrate into the MDA-MB-231 cells and bind specifically with VEGF inside the cells, our anti-VEGF E.Cotector can be said to be perform well and successfully. In the other hand, when our anti-EGFR E.Cotector can bind with EGFR which are displayed on the MCF7 cell surfaces by staining on the surfaces of MCF7 cells.

1. Cultivate MCF7 cell lines and MDA-MB-231 cell lines in 6-well plates. Cultivate three types of E.Coli:

the normal green fluorescent E.Coli, the anti-EGFR E.Cotector, the anti-VEGF E.Cotector for 18 hours and 4mL each.

2. Firstly, remove the cell cultivated solution from the wells. Add in 4% paraformaldehyde to kill and fixed the cells as paraformaldehyde able to prevent the dead cells from floating in the wells. Keep the plates in darkness for 15min.
3. At the same time, separate the LB broth with bacteria pellets by centrifuge. Wash the pellets with PBS buffer and re-dissolve the pellets with PBS buffer.

4. Remove the paraformaldehyde and wash the wells three times with PBS buffer, the time interval is 10min and shake the plates on shaker.

5. Add in triton for permeabilizing the cell membranes and shake in darkness for 10min. Remove triton and repeat the action of washing by using PBS buffer.

6. Add in 500µL of E.Coli in each well. Shake the plates in darkness for 1 hours for cell staining.

7. After 1 hour, remove the E.Coli and wash with PBS buffer. Observe the staining result under the fluorescent microscope.