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.

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.

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.

All our team members have undergone and passed the Chemical, Biological and Fire Safety training requirements from the Office of Safety, Health and Environment (OSHE, http://www.nus.edu.sg/osh/), the department in charge of Laboratory and Work Safety at the National University of Singapore. Furthermore, laboratory specific training has also been given by the respective PIs of the lab where the laboratory work is being carried out (SPS teaching lab at Faculty of Science and A/P M Chang’s Lab at SynCTI). Necessary Hepatitis B vaccinations were also taken in preparation for working with mammalian cell lines.

For each protocol used in our experiments, we have a separate risk assessment. By performing the risk assessment, team members will be aware of the risks involved in the experiment and consequently take appropriate safety precautions. Please refer to our ‘protocols’ page for more information. Our laboratory is equipped with biological and chemical spill kits, and all members of our iGEM Team are trained to handle Biological and Chemical Spills.

The SPS laboratory is classified as Biosafety Level 2, according to the classification by the World Health Organisation (WHO) and the Genetic Modification Advisory Committee of the government of Singapore (http://www.gmac.gov.sg/). The laboratory is controlled by card access which only team members or users that have passed all the safety training requirements are given. All biosafety cabinets, fume hoods and the autoclave have been recertified to be working properly (June 2016–June 2017) by the respective companies and certified inspectors.

Bacterial work and Mammalian cell culture are performed in separate BSL2 Biosafety Cabinets to minimise cross contamination, while manipulation of DNA is done on the open laboratory bench. No cytotoxic reagents are used in our laboratory; SYBR Safe DNA stain for visualisation of DNA is used rather than Ethidium Bromide.

Liquid biological waste is decontaminated using 10% Bleach for 20 min before disposal as normal waste, while solid biological waste are sent for incineration at a local incineration plant devoted to medical waste (Sembcorp http://www.sembcorp.com/en/our-businesses/utilities/on-site-logistics-solid-waste-management).

For the fulfillment of requirements for safety from the iGEM foundation, we have submitted the ‘About our lab’ safety forms (https://2016.igem.org/Safety/About_Our_Lab) and the ‘Final Safety form (https://2016.igem.org/Safety/Final_Safety_Form)

We have also performed a check-in (https://2016.igem.org/Safety/Check_In) for the synthesized invasin gene from Yersinia pseudotuberculosis and the synthesized LLO gene from Listeria monocytogenes.