Biosafety
Considering the central focus of our project, which revolves around the diverse metallo-β-lactamases, it is clear that our top priority was implementing rigorous biosafety measures. The release of enzyme variants with increased activity into the environment could potentially lead to catastrophic consequences. Therefore, biosafety was the primary topic of discussion within the iGEM Bulgaria 2023 team.
Conversations with individuals responsible for instructing us on working with GMOs revealed that human errors are the primary contributing factor to accidental recombinant DNA releases into the environment. To minimize the potential for such incidents within our team, we imposed strict restrictions on working with MBL constructs. These activities were confined to a dedicated laboratory with no windows, access was restricted to only five individuals, and no one was permitted to conduct these experiments alone.
• We tried to use the T7 promotor wherever possible to limit the possibility for expression in case of accidental release.
• We switched to low copy number plasmids like the SC101 ori-based vector pSB4K5 – reducing the overall plasmid numbers also reduce the chance of accidental release.
• We performed regular analyses to confirm that our cell free extracts indeed do not contain any bacterial cells.
Our final and most promising idea was to switch to a new expression host – the yeast Pichia pastoris. In such instances, the constructs must be integrated into the genome, and all associated regulatory sequences should originate from eukaryotic sources. This significantly reduces the chances of gene transfer events to pathogenic bacteria.
We outlined these concepts in a project proposal, which was approved by a non-governmental organization in Bulgaria. As a result, we will receive the necessary financial support for our upcoming host exchange.