Unfortunately, our wet lab only lasted for three months and we were short of time. The completeness of our project was heavily deterred. Inconclusive results from some of the modules have also increases difficulty in demonstrating the full story of B. herculues. Many characterization methods have yet to be attempted. Some experiments could be repeatd as well, and hopefully that would increase confidence in previously obtaiend data.

If more time is allowed, the following would be what we wish to pursue:

1. Finish assembly of the parts from the three modules into pDG1661 and transformed into Bacillus subtilis:
   So far, we have inserted the BMP2 construct (BBa_K733016 and BBa_K733017) and the RPMrel construct (BBa_K733007) into the integration vector pDG1661 separately for characterization. The next step would be to piece these separated parts, as well as the remaining constructs, together to complete our B. herculus. Then we could move on to the final characterization of the strain.
2. Continue the characterization on binding capability 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. When we finished our construct, however, no GFP signal was detected in B. subtilis. One possible reason is that, due to chromosomal integration, only a single copy of GFP coding gene was present per bacterium. Thus the 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 the verification on 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.