Three-month wet lab work is not enough to complete our project and the result we obtained is not conclusive enough to demonstrate the full story of our project. There is still a lot characterization methods need to be kept on testing and a lot of experiments need to be repeated in order to obtain significant data.

1. Finish assembly of the parts from three modules into pDG1661 and transformed into Bacillus subtilis: so far, we have been inserted BMP2 construct (BBa_K733016 and BBa_K733017) and RPMrel construct (BBa_K733007) into vector pDG1661 separately for characterization. The next step is to assemble parts from all three modules into pDG1661, generating our final construct and move on to final characterization.
2. Continue characterizing the binding ability of B. hercules to colon adenocarcinoma HT-29: Originally we plan to co-culture B. hercules with HT-29 cell (colon adenocarcinoma) and HBE16 (Human bronchial epithelial cell) and detect the attached B. subtilis through its fluorescence signal. However, when we finished our construct, we find out that no GFP signal can be detected in B. subtilis. One possible reason is that one single copy of GFP coding gene is not enough to produce detectable amount of GFP. Considering this, we change our characterization plan to visualize bacteria through gram staining. However, since the staining of mammalian cell is not consistence, over-staining and failed destaining happening again and again, we can hardly provide any conclusive evidence to show the binding of B. hercules to tumor cell and provide significant data when comparing with the control group. In this situation, we need to come up with some other more reliable methods to detect the binding.
3. Continue verifying the tumor-apoptosis effect of BMP2 produced by B. hercules. Several rounds of MTT assay has been performed in order to quantify cell proliferation rate after co-culturing mammalian cell with bacteria. However, no matter how gentle we wash after co-culture in order to get rid of bacteria during MTT assay, mammalian cells will detached from the bottom of wells and lose a lot while bacteria are still in contact with mammalian cells. Therefore, other methods need to be proposed in order to characterize the effect of BMP2 to colon cancer cell.