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 increased difficulty in demonstrating the full story of B. hercules. 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. hercules. 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 cells (colon adenocarcinoma) and HBE16 cells (Human bronchial epithelial cells) and then look for attached bacteria through their fluorescence signals. 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 bacterial visualization using Gram stain. Yet, it appeared that not only B. subtilis, but also mammalian cells, could be stained by crystal violet. To make things worse, there was difficulty in achieving 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. A more reliable characterization method is thus necessary.
3. Continue the verification on the tumor-apoptosis effect of BMP2 produced by B. hercules:
   Several rounds of MTT assay have been performed in order to quantify cell proliferation rates after co-culturing mammalian cell with our BMP2 secreting B. subtilis. The technical problem lied in the poor adhension of mammalian cells to the wells, as well as the difficulty in washing B. subtilis off the mammalian cells. A balanced number of washes that could retain the majority of mammalian cells inside the wells while getting rid of B. subtilis was not achievable. Our experience therefore called for another method to characterize the effect of BMP2 on colon cancer cells.