Stabilizing Genetic Programs Against Changes in Context

Synthetic genetic programs often require fine tuning of expression levels to achieve optimal function. They can therefore be quite sensitive to changes in context, as perturbations of gene expression can break this tuning and lead to system failure. To address such fragility, we are creating stabilized promoters that incorporate simple regulatory systems to buffer gene expression against changes in genetic and cellular context. Using TALE proteins as transcriptional repressors in E.coli, we have built a promoter regulated by an incoherent feedforward loop that is nearly insensitive to its copy number (a greater than 20-fold change in gene expression due to copy number is reduced to a less than 50% change in expression). This stabilized promoter allows movement of genetic systems onto most commonly used plasmid backbones with little change in expression. Additionally, it facilitates more predictable gene expression from libraries of random genomic insertions.

The regulatory motifs used in our stabilized promoters are also expected to buffer gene expression against other perturbations, such as a change in the number of free ribosomes or RNAPs within a cell. We are exploring how stabilized promoters buffer gene expression against transient changes in cellular context, including shifts in growth rate due to a media change, or local ribosome depletion caused by protein overexpression. While less striking than the effect on genetic context, the stabilized promoter largely eliminates small perturbations caused by these factors, suggesting that it may yield more predictable gene expression in varying cellular environments. Due to their ability to buffer gene expression against context changes, we hope that stabilized promoters will prove valuable as a foundation for building more robust synthetic genetic programs.