T7 rna polymerase

The sequence of all DNA molecules and expected RNA transcript sequences were chosen to minimize the occurrence of alternative secondary structures checked by the DNA and RNA folding program NUPACK (29 (link)). The DNA and RNA sequences used in this study are listed in Supplementary Section S1. (See Figure S1 for sequence domains and predicted secondary structures.) All DNA oligonucleotides and the short RNA signal iMG were purchased from Integrated DNA Technologies (USA). The T7 RNA polymerase (Cellscript, Madison, WI, USA; #C-AS2607), 10× transcription buffer and thermostable inorganic pyrophosphatase (New England Biolabs, Ipswich, MA, USA; #B9012S, #M0296S), NTP and RNase R (Epicentre, Madison, WI, USA; #RN02825, #RNR07250) were purchased. Malachite Green (MG) dye was purchased from Sigma (#M9015). Since pyrophosphatase is involved in regulating the byproduct inorganic pyrophosphate for our transcriptional circuits and is not directly involved in the dynamics, we neglect this enzyme in our models and do not call it an ‘essential enzyme’ for the circuit dynamics. The nominal concentrations of enzyme stocks quoted by the manufacturer were used: 10.5 μM for RNase R and 6 μM for T7 RNA polymerase (RNAP).