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

Abstract

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 glycineserine-glycine (GSG) prefix or by using two different 2As sequences in tandem. Remarkably, we finally realized a well-working tricistronic 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.

Laccase is a multitasking protein for synthetic gene circuits in the yeast Saccharomyces cerevisiae

“…Each of the other two systems demanded to assemble two new TUs. One TU was for TtLcc1 expression under the control of a synthetic regulated promoter, either 2xlex2Op_pCYC1core [ 25 ] or pCYC1min_tetOp [ 26 ] (see Fig. 3 A and S3B-D).…”

Design and engineering of logic genetic-enzymatic gates based on the activity of the human CYP2C9 enzyme in permeabilized Saccharomyces cerevisiae cells

“…More precisely, both CYP2C9 and CPR were placed in the first position of a bi-cistronic cassette upstream of the EBRV-1 2A peptide sequence and yEGFP. EBRV-1 was previously reported to have a high cleavage efficiency in eukaryotic cells [ 21 ]. The two bi-cistronic sequences were flanked, initially, by the strong yeast constitutive GPD promoter (pGPD) and the CYC1 terminator (CYC1t).…”

“…A DNA sequence encoding for the 2A peptide from the equine rhinitis B virus-1 (EBRV-1) was used to construct two synthetic S. cerevisiae bicistronic transcription units (TUs) where either the human CPR or CYP2C9 gene was linked to the yeast enhanced green fluorescence protein ( yEGFP ) [ 20 , 21 ]. Strains successfully transformed with the two TUs were characterized via fluorescence measurements and then permeabilized using 0.3 % concentration of Triton X-100.…”

The 2A Story: The End of the Beginning

“…In the case of shorter 2As, cleavage efficiency has been improved by insertion of various spacer sequences immediately upstream of the 2A sequence such as a glycine-serine linker (e.g. -GSG-or -SGSG-, [27][28][29]; a 3XFLAG epitope tag -DYKDHDG-DYKDHDI-DYKDDDDK-, [30]; or a V5 epitope tag -GKPUPNPLLGLDST- [31]. These "flexible" linkers create a space between the N-terminal protein and the 2A peptide, favouring a conformation which facilitates efficient cleavage [32].…”