Genetic circuit design automation for yeast

Abstract

Cells can be programmed to monitor and react to their environment using genetic circuits. Design automation software maps a desired circuit function to a DNA sequence, which requires units of gene regulation (gates) that are simple to connect and behave predictably. This poses a challenge for eukaryotes due to their complex mechanisms of transcription and translation. To this end, we have developed gates for yeast (Saccharomyces cerevisiae) that are connected using RNA polymerase (RNAP) flux as the signal carrier and are insulated from each other and host regulation. They are based on minimal constitutive promoters (~120bp), for which rules are developed to insert operators for DNA-binding proteins. Using this approach, we construct nine NOT/NOR gates with nearly-identical response functions and 400-fold dynamic range. In circuits, they are transcriptionally insulated from each other by placing ribozymes downstream of terminators to block nuclear export of mRNAs resulting from RNAP readthrough. Based on these gates, Cello is used to build circuits to specification with up to 11 regulatory proteins. A simple dynamic model predicts the circuit response over days. Extending genetic circuit design automation to eukaryotes simplifies their incorporation into cellular engineering projects, whether it be to stage processes for bioproduction, serve as environmental sentinels, or guide living therapeutics.

Robust and flexible platform for directed evolution of yeast genetic switches

“…Among them, the performance of the DAPG-ON switch was comparable to that of the Tet-ON switch, which is known as one of the best switches in terms of its fold induction (>10 2 ) ( Fig. 5b), as well as that of the recently reported high-performing switches 75 . We then demonstrated that these evolved switches could be further improved in fold induction simply by tweaking the numbers of operators 10 tandemly aligned in synPs.…”

“…Evidently, adding this sequence to p tetO7 caused significant leakage of the GFP expression ( Supplementary Figure 18). A set of terminators and ribozyme sequences could be used to insulate synPs from their upstream sequences to block their transcription read-through 75 .…”

Metabolite trafficking enables programmed terpene secretion by yeast

“…However, this could be mitigated by balancing or tuning the expression levels of relevant proteins. Furthermore, synthetic regulatory systems with diverse operational modes 31,32 , such as auto-regulatory feedback loops, toggle switches, and engineered riboswitches, may permit the decoupling or integration of cellular growth and terpene secretion.…”

Engineering Bacterial Cellulose by Synthetic Biology

“…The rich information available at the genome, transcriptome, proteome and metabolome levels for these organisms have made them the go-to cells for creating modular DNA part libraries and rapid cloning toolkits that can quickly and easily combine these DNA parts into novel genetic programs [37,38]. Synthetic biology projects in these microbes regularly now involve the design and construction of synthetic DNA programs 10 s and 100 s of kilobases in length, often containing dozens of modular DNA parts and typically with many design variants built and tested in parallel [39,40].…”