Introduction:
Colorectal cancer is the second most deadly cancer worldwide. Current screening methods have low detection rates and frequently provide false positive results, leading to missed ...diagnoses or unnecessary colonoscopies. To tackle this issue, the Wageningen UR iGEM team from 2022 developed “Colourectal”, a living diagnostic tool for colorectal cancer. Following a synthetic biology approach, the project used an engineered
Escherichia coli
Nissle 1917 strain capable of binding to tumour cells that detects two distinct cancer biomarkers, and secretes a coloured protein observable in stool. Due to the utilization of genetically modified bacteria
in vivo
, precautionary biosafety measures were included within a three level safe-by-design strategy.
Results:
The first genetic safeguard ensured confinement of the living diagnostic to the colon environment by implementing auxotrophy to mucin that is abundant in the colon lining. For this, a synthetic chimeric receptor was generated to ensure expression of essential genes in the presence of mucin. The second strategy limited the viability of the engineered bacteria to the human body, preventing proliferation in open environments. The use of a temperature sensitive kill switch induced bacterial cell death at temperatures below 37°C. The third biocontainment strategy was installed as an emergency kill switch to stop the Colourectal test at any point. By inducing a highly genotoxic response through CRISPR-Cas-mediated DNA degradation, cell death of
E. coli
Nissle is triggered.
Discussion:
While the use of engineered microorganisms in human applications is not yet a reality, the safety considerations of our multi-layered strategy provide a framework for the development of future living diagnostic tools.
Synergistic and supportive interactions among genes can be incorporated in engineering biology to enhance and stabilize the performance of biological systems, but combinatorial numerical explosion ...challenges the analysis of multigene interactions. The incorporation of DNA barcodes to mark genes coupled with next-generation sequencing offers a solution to this challenge. We describe improvements for a key method in this space, CombiGEM, to broaden its application to assembling typical gene-sized DNA fragments and to reduce the cost of sequencing for prevalent small-scale projects. The expanded reach of the method beyond currently targeted small RNA genes promotes the discovery and incorporation of gene synergy in natural and engineered processes such as biocontainment, the production of desired compounds, and previously uncharacterized fundamental biological mechanisms.
