Part:BBa_K1202114:Experience

-The Experiments about EpCAM – Anti EpCAM Binding Firstly we wanted to test that can our bacteria produce C215 (Anti-EpCAM). To test this, we did Immunofluorescence experiments. We put OmpA (a signal peptide which send proteins to membrane) signal peptide sequence and his-tag sequence before C215 protein sequence in our one part. And then we did immunofluorescence and we waited to see a red circle around our bacteria and we saw. We added his-tag because C215 was staining thank to his-tag. During the experiments we used anti-his-tag, after than the substance which will paint… At this step we understood that our bacteria can produce C215 because we saw red circles. The second, we did another immunofluorescence experiment too. This experiment’s purpose was to understand that will EpCAM and our C215 bind? Because our experiment which the other immunofluorescence (the OmpA was using there) experiment, we understood that bacteria is producing C215, but we couldn’t understand anything about binding. This time we used cancer cells and we didn’t use bacteria, we used purified C215 to take some information about binding. We put our purified proteins in well-plates with cancer cells. We were expecting to see red color circle around cancer cells and to seeing cancer cells’ nucleus blue.

Immunofluorescence c215 In our project we need that our anti-EpCAM active domain c215 bind the membrane of cancer cells. So we decided to make immunofluorescence experiment for tested that our c215 is binding or not? As you see in Pic. 1 the little red shining cells are our results and show that our c215 is working. With this results we saw that our nanofactory will probably bind to our cancer cells successfully. Immunofluorescence OmpA Before we start to made experiments we decided to use an alternative methods for detection cancer cells (Nano factory). We decided to use our bacteria as a nanofactory (quorum sensing). We added OmpA in front of our c215 so our bacteria can bind cancer cells. So we tested OmpA that produced by our bacteria and we made immunofluorescence experiments. So as a results see in Pic 2 (little red shining bacteria) our OmpA presented by our cells to outer membrane successfully. So that means we will use our bacteria as a Nanofactory.

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