Cell-Free Synthetic Biology System

IVT2H reagents are described previously20 (link)21 (link)22 . Briefly, IVT2H contained 144 nM purified E. coli RNA polymerase core enzyme, 1.2 μM purified recombinant E. coli IHF, 0.8 units/μl murine RNase inhibitor, the PURExpress^®in vitro protein synthesis system (New England Biolabs), 0.2 ng/μl (45 pM) plasmid DNA expressing σ54, 4.4 nM linear reporter DNA expressing GFP and 0.2 ng/μl (60 pM) plasmid DNA expressing hybrid fusion protein AD-MDM2, in which the activation domain (AD, residues 1–296) of PspF was fused to the full-length human MDM2 protein. The genes for the wild-type p53 peptide (p53p, residues 17–26: ETFSDLWKLLPE) and the peptide inhibitor (PMI: TSFAEYWNLLSP) were synthesized and fused to the N-terminus of the DNA binding domain Cro (DB). The linear DNA constructs expressing p53p-DB, PMI-DB or the PMI library-DB were used in drop-based microfluidics experiments.