Considering the requirements of our project, Bacillus subtilis, a gram positive model organism in lab study, is chosen to be the chassis in our project. Its ideality can be illustrated in the following aspect:

1. Safety

Synthetic biology is always challenged on its safety issues especially when it comes to the area of medical treatment. From bacteria host to the final bio-waste, worries raised from the public have never been released. In order to ease the concern from the public, researchers try their best to minimize the hazards from each step of experiments. Regarding safety issues, We, 2012 HKUST iGEM team, there choose to use B. subtilis, a non-pathogenic chassis.

1. Low degree of virulence:

Inhabiting gut as a part normal flora, Bacillus subtilis is considered to be a non-pathogenic organism to human. Rarely has infection case in human been revealed resulting from B. subtilis. According to 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 the other word, B. subtilis possess low virulence to human and presents low risk of adverse effects to human health.  

2. Establishment of Integration vectors:

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 purpose. In our project, we employed integration vector not only because of its stability in B. subtilis but also because of its advantage 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 tissue can induce adverse effect, integrating target gene into genome can reduce the chance of spreading of BMP2 gene through horizontal transformation and avoid non-specific drug release in gut.

2. Protein secretion:

Comparing with E. coli, another commonly used chassis in iGEM, Bacillus subtilis is preferred because of its high capacity in secreting proteins to extracellular environment. As a gram positive eubacteria, B. subtilis lacks of outer membrane. Only with a layer of 10 to 50nm peptidoglycan outside cytoplasmic membrane, Bacillus subtilis can secrete protein directly into extracellular environment. Since we designed to have bacteria producing while secreting BMP2 out to colon, this property right meets our requirement. 

3. Peptide displaying:

Lacking the outer membrane, Bacillus subtilis is ideal for peptide displaying. As the outer most layer of B. subtilis, its cell wall 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. (217) Aiming to display the tumor-binding peptide, RPMrel, on bacteria surface, we decide 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.

4. Normal flora in gut:

Edberg, S.C. 1991. US EPA human health assessment: Bacillus subtilis. Unpublished, U.S. Environmental Protection Agency, Washington, D.C.
217. Pooley, H. M., R. Merchante, and D. Karamata. 1996. Overall protein content and induced enzyme components of the periplasm of Bacillus subtilis. Microb. Drug Resist. 2:9–15.