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:
   We planned to co-culture green fluorescence labelled B. hercules with HT-29 cell (colon adenocarcinoma) and HBE16 (Human bronchial epithelial cell) and then detect attached B. subtilis through its fluorescence signal. The labelled B. subtilis was constructed but no GFP signal could be detected. Since the GFP coding gene was integrated into the genome as a single copy, inadequate expression might had yielded undetectable signal. As an alternative, we turned to visualizing bacteria through applying Gram stain. It appeared that not only B. subtilis, but also mammalian cells, could be stained by crystal violet. To make things worse, there was difficulty to achieve consistent staining in mammalian cells, since they were often over-stained or poorly destained. Conclusive evidence, with respect to adhensions between B. hercules and tumor cells, could not be produced from the much limited method. In view of the situation, more reliable characterization methods are needed.
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.