We chose Bacillus subtilis - a model Gram-positive bacterial species often used in laboratory study - as our chassis. It is ideal as a base for designing mechanisms to fulfill our project aims for reasons we mention below.

Safety

The safety issues concerning synthetic biology are numerous, particularly when it comes to the area of medical treatment. To reduce the chance of recombinant bacteria causing harm, synthetic biologists try to develop and utilize solutions that minimize the potential hazards of their products. Regarding safety issues, we the HKUST iGEM 2012 team chose to use B. subtilis, a non-pathogenic chassis.

Inhabiting gut as a part of normal flora, Bacillus subtilis is considered to be a non-pathogenic organism to human. There is few case that human beings are infected by B. subtilis. According to Edberg (Edberg 1991), B. subtilis does not produce significant quantities of extracellular enzymes or possess other virulence factors that would predispose it to cause infection. In other word, B. subtilis possesses low virulence to human and presents low risk of adverse effects to human health.  

The discovery of natural integration in Bacillus subtilis raised great attention in the field of molecular genetics in 1978. Due to its natural integration, a series of integration vectors for B. subtilis were designed later for different purposes. In our project, we employed an integration vector not only for its stability in B. subtilis, but also its advantages in safety. The employment of integration vector to some extent minimizes the risk of antibiotic resistance spreading within the normal flora in gut. In addition, since the exposure of BMP2 to normal tissues can induce adverse effect, integrating target genes into genome can reduce the chance of spreading of Bmp2 gene through horizontal gene transfer and avoid non-specific drug release in gut.

Comparing with E. coli, another commonly used chassis in iGEM, Bacillus subtilis is preferred because of its high ability in secreting proteins to extracellular environment. As a Gram-positive eubacteria, B. subtilis lacks an outer membrane, only with a layer of 10 to 50nm peptidoglycan outside cytoplasmic membrane. Thus Bacillus subtilis can secrete proteins directly into the extracellular environment more easily. Since we are aiming for bacterial secretion of BMP2 out to the colon, this property suits our purposes well. 

Without the outer membrane, Bacillus subtilis is ideal for peptide displaying. The cell wall of B. subtilis is the surface of the bacterium and contains approximately 9% of the total protein. The discovery and study in cell wall binding modules from cell wall bound proteins provides an attractive tool for surface display of peptide. (Pooley et al. 1996) Aiming to display the tumor-binding peptide, RPMrel, on bacteria surface, we decided to take advantage of the cell wall displaying system, LytC, in Bacillus subtilis. Thus, the ease of peptide displaying serves as one of our reasons to choose B. subtilis as our chassis.

B. subtilis not only exists widely in the nature, but also contributes to part of the normal flora in gut. (Collin and Gibson 1999) Regarded as a probiotic, it has been proved to have positive effects on patients suffering from gaseous symptom (Corazza et al. 1992). Using B. subtilis as our chassis, in theory, will not introduce any exotic species into gut either.

References:
Edberg,S.C.. 1991. US EPA human health assessment: Bacillus subtilis. Unpublished, U.S. Environmental Protection Agency, Washington, D.C.
Pooley H. M., Merchante R. and Karamata D.. 1996. Overall protein content and induced enzyme components of the periplasm of Bacillus subtilis. Microb. Drug Resist.2:9–15.
Collins M. D. and Gibson G. R.. 1999. Probiotics, prebioticsand synbiotics: Approaches for modulating the microbial ecology of the gut. Am. J. Clin. Nutr. 69:1052S–1057S
Corazza G.R., Benati G., Strocchi A., Sorge M. & Gasbarrini G.. 1992. Treatment with Bacillus subtilis reduces intestinal hydrogen production in patients with gaseous symptoms. Current therapeutic research. 52.1: 144-151