T7Max transcription system

Abstract

Efficient cell-free protein expression from linear DNA templates has remained a challenge primarily due to template degradation. Here we present a modified T7 RNA polymerase promoter that acts to significantly increase the yields of both transcription and translation within in vitro systems. The modified promoter, termed T7Max, recruits standard T7 RNA polymerase, so no protein engineering is needed to take advantage of this method. This technique could be used with any T7 RNA polymerase- based in vitro protein expression system. Unlike other methods of limiting linear template degradation, the T7Max promoter increases transcript concentration in a T7 transcription reaction, providing more mRNA for translation.

Akaby—Cell-free protein expression system for linear templates

“…In that plasmid, BamHI cuts at position 182 nt after the eGFP coding sequence, thus linearizing the eGFP plasmid without affecting the open reading frame. To further study the robustness of the system, we used eGFP templates under two different T7 RNA polymerase promoters, the canonical T7 promoter and the new high yield T7Max promoter [ 22 ]. Because TXTL components generate a low level of endogenous fluorescence in the green channel, we used a no template control (NTC) as the background fluorescence benchmark.…”

Expanding luciferase reporter systems for cell-free protein expression

“…1C) 22,24 . To test their activities in TXTL, we individually cloned H3H, Luz, LuxA, LuxB, and Fre genes into a vector plasmid designed for T7 RNA polymerase-coupled TXTL expression 25 . We confirmed that both Luz-H3H and LuxAB-Fre systems generate luminescence (Fig.…”

“…A plasmid for fungi luciferase pathway, P307-FBP_6, was a gift from Daniel Voytas lab at the University of Minnesota 20 . Cloning vector plasmids, pCI-T7Max-UTR1-CTerminus8xHis-T500 and pCI-T7Max-UTR1-NTerminus8xHis-T500, were obtained from our lab stock 25 . Plasmids for other luciferases, pGreen_dualluc_3’UTR_sensor, pGEN-luxCDABE, pUAS-NanoLuc, and pBad-LgBiT-PhoCl1-SmBiT-MBP, were purchased from Addgene 28– 30 .…”

“…S10 ). First, we cloned NPGA, Hisps, and CPH genes into a vector plasmid, pCI-T7Max-UTR1-CTerminus8xHis-T500 or pCI-T7Max-UTR1-NTerminus8xHis-T500 25 . Those genes’ expressions were confirmed by Western blot ( Fig.…”

Parasites, infections and inoculation in synthetic minimal cells

“…We used a strong T7 RNAP promoter T7Max, which demonstrates significantly higher transcription yield, resulting in correspondingly higher translation yields, in TxTl. 17 We tested the reaction setup identical to the one described earlier: two plasmids at different ratios, tested at varying final total plasmid concentration. One of the plasmids was under the control of the strong T7Max promoter, while the other was under the canonical T7 promoter.…”