Host cell response to synthetic biology: a deeper look for more robust and optimised designs

Gene expression in living cells requires the use of host cell resources and precursors, like polymerases, ribosomes, amino acids and nucleotides. Synthetic biology systems thus place de facto a “load” on their host cells, which, in turn, causes a change in cell fitness and growth rate. Researchers are now becoming aware that this loading effect can lead to unpredictable interactions and unwanted behaviours that characterise many of the failures in synthetic biology, such as inactivating mutations.

Here, we present results pertaining to the characterisation of the burden caused by a library of synthetic biology devices embedded in two different E. coli strains. For this characterisation, a novel “capacity monitor” genetic device is used as a tool to quantify the burden imposed by synthetic gene expression via the measurement of both heterologous gene expression and host cell capacity with a standardised, plate-based assay that uses flow cytometry for single cell analysis.

Based on this method for burden quantification, we identify a variety of synthetic devices and pathways that cause significant burden to cells, and use quantitative PCR to reveal some native genes that significantly modulate their expression within the first hour of burden induction. These native genes represent potentially useful biomarkers of the native burden/stress. To understand and exploit this native burden response at a deeper level, transcriptome analysis of burdened cells is now in progress using RNAseq with the aim of identifying native regulators that can be repurposed in future burden-based feedback genetic devices.