Design of Adjacent Transcriptional Regions to Tune Gene Expression and Facilitate Circuit Construction

Polycistronic architecture is common for synthetic gene circuits, however, it remains unknown how expression of one gene is affected by the presence of other genes/noncoding regions in the operon, termed adjacent transcriptional regions (ATR). Here, we constructed synthetic operons with a reporter gene flanked by different ATRs, and we found that ATRs with high GC content, small size, and low folding energy lead to high gene expression. Based on these results, we built a model of gene expression and generated a metric that takes into account ATRs. We used the metric to design and construct logic gates with low basal expression and high sensitivity and nonlinearity. Furthermore, we rationally designed synthetic 5'ATRs with different GC content and sizes to tune protein expression levels over a 300-fold range and used these to build synthetic toggle switches with varying basal expression and degrees of bistability. Our comprehensive model and gene expression metric could facilitate the future engineering of more complex synthetic gene circuits.