Adjusted Experimental Procedures in light of safety issues
Issue: Ethidium Bromide
Solution: Hydragreen (non-toxic)
We avoided the use of ethidium bromide, a DNA intercalator / mutagen, when staining all of our gels, in order to prevent potential DNA damage when handling. Instead, our laboratory decided to use HydraGreen which has been determined as safe for handling in the laboratory. Regardless, when handling gels, gloves were worn at all time.
CHLOROFORM EXTRACTION OF PERIPLASMIC PROTEIN
Issue: Chloroform extraction
Solution: High safety and focusing on insulin production without requiring cell lysis.
As chloroform is extremely toxic and can have severe effects on the central nervous system. However, as chloroform is the only well established method for periplasmic protein extraction from our research ,we compromised its usage by increasing the stringency of our laboratory safety and minimised our usage. Minimal quantities of chloroform were used where possible and the entire USYD iGEM team were made aware of all the safety issues via safety forms, prior to handling the chemical.
Due to the problems of chloroform for extraction of periplasmic insulin, our team focused on optimising Bacillus subtilis insulin production as it involved secretion into the media. Purification of insulin in Bacillus does not involve the need to lyse cell walls and only removal of media. This method would additionally be commercially ideal, particularly as our project design is to minimise the need for additional purification steps. The lack of need for chemicals like chloroform will have significant benefits, especially in large scale insulin production.
Issue: Acrylamide and other toxic substances
Solution: Pre-Cast Gels
Preparation of SDS-PAGE gels involved several chemicals which were not ideal in the laboratory including hydrochloric acid which is corrosive, but primarily acrylamide. Acrylamide is a carcinogenic substance and a mutagen which can cause damage even when inhaled. Due to this danger, not only to the person working with the acrylamide, but others in the laboratory, we decided to avoid making our own SDS-PAGE gels and instead ordered pre-cast gels. These gels are packaged and sealed and were only opened when needed, minimising the exposure of acrylamide to the laboratory as we required many of them to analyse our cell lysates for upregulated insulin production.
RADIOACTIVE 14C IN HEPATOCYTE ASSAY
Issue: Use of 14C radioactive carbon to measure insulin bioactivity
Solution: Supervision and training
We tested the bioactivity of insulin by measuring the cell's uptake of radioactive glucose. Importantly, this is the 'gold standard' to test insulin in human and mouse cell lines so we decided to still use the assay instead of an alternative. We did however make sure we performed an ELISA first and only used successful results to ensure we minimised contact with radioactive material.
Most importantly, we were supervised throughout the entire procedure by researchers who were experienced in these cell assays. Additionally, we used the smallest amount of radioactive glucose for our results to reduce any risks.