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.

Low degree of virulence.

As a natural member of the human gut microbiome, B. subtilis is considered to be non-pathogenic to humans. There are very few cases of humans being infected by B. subtilis, and of those who have been infected the vast majority had severe immune deficiency. 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 humans and has a low risk of adverse effects to human health.

Establishment of integration vectors.

The discovery of natural integration in B. subtilis raised great attention in the field of molecular genetics in 1978. A series of integration vectors for B. subtilis were designed later for different purposes. For our project we employed an integration vector not only for its stability in B. subtilis, but also its advantages in safety. The employment of the 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 effects, integrating target genes into the genome can reduce the chance of spreading the Bmp2 gene through horizontal gene transfer and avoid non-specific drug release in gut.

Protein secretion.

Compared with E. coli, another commonly used chassis in iGEM, B. subtilis is preferred because of its reliability in secreting proteins directly to the extracellular environment. As a Gram-positive eubacteria, B. subtilis lacks an outer membrane, featuring only a 10-50nm peptidoglycan layer beyond its the plasma membrane. Since we are aiming for bacterial secretion of BMP2 out into the digestive tract in the vicinity of colon cancer cells, this property suits our purposes well.

Peptide display.

Without an outer membrane, B. subtilis is ideal for displaying items on its surface. The cell wall of B. subtilis is the surface of the bacterium and contains approximately 9% of the total protein in the cell. The discovery and study of cell wall binding devices produced 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 display system, LytC, in B. subtilis. Thus, the ease of peptide display serves as one of our reasons to choose B. subtilis as our chassis.

Part of gut commensal flora.

B. subtilis does not only exist widely in nature, but also contributes to part of the normal flora in the gut (Collin and Gibson 1999). Regarded as a probiotic, it has been proved to have positive effects on patients suffering from functional abdominal bloating (Corazza et al. 1992). Using B. subtilis as our chassis will in theory mean we will not be introducing any exotic species into the gut.

References.

Collins M. D. and Gibson G. R.. 1999. Probiotics, prebiotics and 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

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.