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 aspects:

1. Safety

Safety issues concerning synthetic biology are always challenged, especially when it comes to the area of medical treatment. From bacteria hosts to biowastes, the public has never ceased to raise worries. To ease concerns from the public, researchers try their best to minimize biohazards involved in every step of their experiments. Regarding safety issues, we, 2012 HKUST iGEM team, therefore chose 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 (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 purposes. In our project, we employed an 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 genes into genome can reduce the chance of spreading of BMP2 gene through horizontal gene transfer 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 an outer membrane and only a layer of 10 to 50nm peptidoglycan outside cytoplasmic membrane. Thus Bacillus subtilis can secrete proteins directly into the extracellular environment. Since we were aiming for bacterial secretion of BMP2 out to the colon, this property suits our purposes well. 

3. Peptide displaying:

Lacking 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 served as one of our reasons to choose B. subtilis as our chassis.

4. Normal flora in gut:

B. subtilis not only exists widely in the nature, but also contributes to part of the normal flora in gut. (Collon M.D. & Gibson G.R.) Regarded as a probiotic, it has been proved to have positive effects on patients suffering from gaseous symptom (Corazza1 et al). Using B. subtilis as our chassis, in theory, will not introduce any exotic species into gut as well.

Reference:
Edberg, S.C. 1991. US EPA human health assessment: Bacillus subtilis. Unpublished, U.S. Environmental Protection Agency, Washington, D.C.
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.
Collins, M. D., and G. R. Gibson. 1999. Probiotics, prebioticsand synbiotics: Approaches for modulating the microbial ecology of the gut. Am. J. Clin. Nutr. 69:1052S–1057S
Corazza1 G.R., G. Benati, A. Strocchi, M. Sorge & G. Gasbarrini. 1992. Treatment with Bacillus subtilis reduces intestinal hydrogen production in patients with gaseous symptoms. Current therapeutic research. 52.1: 144-151