Logic Circuits Based on 2A Peptide Sequences in the Yeast Saccharomyces cerevisiae

Gene digital circuits are the subject of many studies in Synthetic Biology due to their various applications from pollutant detection to medical diagnostics and biocomputing. Complex logic functions are calculated via small genetic components that mimic Boolean gates, i.e., they implement basic logic operations. Gates interact by exchanging proteins or noncoding RNAs. To carry out logic operations in the yeast Saccharomyces cerevisiae, we chose three bacterial repressors commonly used for proofs of concept in Synthetic Biology, namely, TetR, LexA, and LacI. We coexpressed them via synthetic polycistronic cassettes based on 2A peptide sequences. Our initial results highlighted the successful application of four 2A peptides─from Equine rhinitis B virus-1 (ERBV-1 2A), Operophtera brumata cypovirus 18 (OpbuCPV18 2A), Ljungan virus (LV2A), and Thosea asigna virus (T2A)─to the construction of single and two-input Boolean gates. In order to improve protein coexpression, we modified the original 2A peptides with the addition of the glycine-serine-glycine (GSG) prefix or by using two different 2As sequences in tandem. Remarkably, we finally realized a well-working tri-cistronic vector that carried LexA-HBD(hER), TetR, and LacI separated, in the order, by GSG-T2A and ERBV-1 2A. This plasmid led to the implementation of three-input circuits containing AND and OR gates. Taken together, polycistronic constructs simplify the cloning and coexpression of multiple proteins with a dramatic reduction in the complexity of gene digital circuits.