Coupled in vitro transcription/translation based on wheat germ extract for efficient expression from PCR-generated templates in short-time batch reactions

Cell-Free Biosensors Based on Modular Eukaryotic Riboswitches That Function in One Pot at Ambient Temperature

“…The features are that a terminal 5′ GAA trinucleotide on mRNA functions similar to the 5′ cap, while a 5′ GGG trinucleotide at the terminus (especially in a duplex) effectively prevents ribosome loading. , Because the aptazyme already has a duplex with 5′ GGG, 5SL, at the 5′ terminus and was designed in advance so that the 3′-side cleaved fragment has the terminal 5′ GAA after self-cleavage, we needed only to add at the 3′ terminus a gene (an open reading frame) encoding a reporter fluorescent protein (YPet) with a 5′ enhancer named E01 and an 1183-nt 3′ UTR (Figure S2A). When the DNA template of this riboswitch candidate, RS-c1-theo , was incubated for 3 h at 26 °C in the absence or presence of 1 mM theophylline in a WGE-based coupled in vitro transcription/translation (cIVTT) system containing T7RNAP in the SUB-AMIX buffer, it showed a moderately high ON/OFF induction ratio (approximately 12) (Figure S2D). However, although the expression efficiency under the OFF condition (i.e., without theophylline) was sufficiently low, comparable to that of a CM-free and thus cleavage-free OFF control (c1-OFF-ctrl, Figure S2A), the expression efficiency under the ON condition (i.e., with 1 mM theophylline) was approximately 40-fold lower than that by a 50-fold larger amount of an aptazyme-free positive control mRNA (PC-mRNA, Figure S2C) under the same WGE-based cIVTT conditions.…”

“…The latter three components (CK, SUB-AMIX, and WEPRO1240) were part of the WEPRO1240 Expression Kit (CellFree Sciences). The mixture was then incubated at 26 °C for 3 h. The translation efficiency was evaluated using either the fluorescence intensity of translated YPet or the chemiluminescence intensity of translated nLuc, as previously described. , …”

“…The mixture was then incubated at 26 °C for 3 h. The translation efficiency was evaluated using either the fluorescence intensity of translated YPet or the chemiluminescence intensity of translated nLuc, as previously described. 24,39 ■ ASSOCIATED CONTENT * sı Supporting Information…”

“…cIVTT was performed in WGE under previously optimized conditions with slight modification. 39 A total of 10 μL of a mixture was prepared, consisting of a 10 nM dsDNA template or a 500 nM mRNA template, various concentrations of analyte molecules, 2.5 U/μL T7RNAP, 0.375 mM of each NTP (1.5 mM NTPs) except those in SUB-AMIX, 1.5 mM MgCl 2 , 40 ng/μL creatine kinase (CK), 1× SUB-AMIX (containing 2.7 mM Mg(OAc) 2 , 1.2 mM ATP, and 0.25 mM GTP), and 20 v/v% WEPRO1240 (WGE). The latter three components (CK, SUB-AMIX, and WEPRO1240) were part of the WEPRO1240 Expression Kit (CellFree Sciences).…”