Team:NUS Singapore/Safety
Safety in our Project
Safety is imperative for the smooth functioning and progress of our project, especially since it involves the use of a variety of biological agents. Therefore, the team views safety very seriously and has put in much effort to ensure all protocols are up to standard. Biosafety in our project involves minimising the risks to the researchers working in the laboratory, as well as the general public in future medical applications based on our research. In line with our emphasis on safety in the laboratory, we are proud to announce that we have obtained an ‘A’ grade for the annual Faculty of Science Housekeeping Inspection 2016 for lab safety over the summer of 2016, an achievement for two consecutive years. We endeavour to continue maintaining a safe working environment for all researchers.
Safety when handling biological organisms
Non-pathogenic strains of E. coli such as BL21 and DH5α from Life Technologies, NEB Stable Competent E. coli and Nissle 1917 Δalr ΔdadX were used for cloning of plasmids and expression of proteins of interest. These strains are Risk group 1 and were handled in a BSL2 Biosafety cabinet. The E. coli strain carrying the invasin and listeriolysin O (LLO) was handled as a Risk Group 2 agent. Mammalian human cell lines, HeLa, HepG2, MDA-231 and MDA-468 are classified under Risk group 2 and were also handled in a BSL2 Biosafety cabinet. Training for handling of mammalian cells or any new experimental protocol was provided by the principal investigator (PI) to all team members prior to the experiments.
Safety in Project Design
In our project, we aimed to engineer E. coli as a flexible vector that has the potential to specifically detect solid tumours using spatial markers intrinsic to the cancer. One intrinsic property that we are utilising is the elevated lactate concentrations around the solid tumour and hence the E. coli are engineered to only survive in areas of high lactate by placing the gene that produces alanine racemase (a pertinent enzyme to convert L-alanine to D-alanine and hence important for E. coli survival) under a lactate-sensitive promoter (BBa_K1847008 and BBa_K822000). Hence, without the presence of elevated concentrations of lactate, the gene will not be expressed and the engineered E. coli will not survive. Next, in order for our engineered E. coli to enter the mammalian cells, we used the synthesized invasin gene from Yersinia pseudotuberculosis and the LLO gene from Listeria monocytogenes. The invasin protein allows for bacteria to enter mammalian cells via β1-integrin, while LLO is a pore-forming protein that enables the bacteria to escape the endosome. These two proteins are part of the pathogenicity island of their respective species. The invasin and LLO proteins enable our engineered E. coli to enter mammalian cells, and escape the endosome, where they can potentially deliver an encoded therapeutic to kill the tumour cell. These two proteins contribute to the pathogenicity of the bacteria, thus precaution or control must be in place to minimise any unwanted expression when there is accidental contact with normal cells.To ensure that these proteins are only expressed under the conditions of the tumour microenvironment, the invasin and LLO proteins will be placed under the control of the has operon and gene expression can only be activated via a quorum sensing system.
Financial Strategy
To fund our operations, NUS_Singapore has received a start-up budget from the National University of Singapore Faculty of Science Dean’s Office and will be supplementing additional costs with applications to a range of grants that support start-ups such as the ACE Startups grant offered by SPRING Singapore. Each use of RIOTSystem as a diagnostic is projected to be sold at SGD$100, a competitive price as compared to other diagnostic kits and tests currently offered in Singapore for cancer detection as those are in the range of SGD$50-$1000. Furthermore, we have made a modest estimate of 3000 units sold within the first year of establishment based on the receptiveness of the participants of our preliminary survey. This will give us a net projected income of $181,711.26 before tax and the provision of salary. This figure is projected to rise in subsequent years because the RIOTsystem is upscalable at lower cost and some of the fixed costs incurred are one-off payments, such as registration fees for the company.
Marketing Strategy
TBA
