scholarly journals Synthetic logic circuits using RNA aptamer against T7 RNA polymerase

2021 ◽  
pp. 2000449
Author(s):  
Jongmin Kim ◽  
Juan F. Quijano ◽  
Jeongwon Kim ◽  
Enoch Yeung ◽  
Richard M. Murray
Keyword(s):  
Rna Polymerase ◽  
Logic Circuits ◽  
T7 Rna Polymerase ◽  
Rna Aptamer

scholarly journals Synthetic logic circuits using RNA aptamer against T7 RNA polymerase

2014 ◽  
Author(s):  
Jongmin Kim ◽  
Juan F Quijano ◽  
Enoch Yeung ◽  
Richard M Murray
Keyword(s):  
Rna Polymerase ◽  
Expression System ◽  
Building Blocks ◽  
Logic Circuits ◽  
T7 Rna Polymerase ◽  
Free Expression ◽  
Rna Aptamer ◽  
Cell Free Expression System

Recent advances in nucleic acids engineering introduced several RNA-based regulatory components for synthetic gene circuits, expanding the toolsets to engineer organisms. In this work, we designed genetic circuits implementing an RNA aptamer previously described to have the capability of binding to the T7 RNA polymerase and inhibiting its activity in vitro. Using in vitro transcription assays, we first demonstrated the utility of the RNA aptamer in combination with programmable synthetic transcription networks. As a step to quickly assess the feasibility of aptamer functions in vivo, a cell-free expression system was used as a breadboard to emulate the in vivo conditions of E. coli. We tested the aptamer and its three sequence variants in the cell-free expression system, verifying the aptamer functionality in the cell-free testbed. In vivo expression of aptamer and its variants demonstrated control over GFP expression driven by T7 RNA polymerase with different response curves, indicating its ability to serve as building blocks for both logic circuits and transcriptional cascades. This work elucidates the potential of RNA-based regulators for cell programming with improved controllability leveraging the fast production and degradation time scales of RNA molecules.

scholarly journals Revisiting T7 RNA polymerase transcription in vitro with the Broccoli RNA aptamer as a simplified real-time fluorescent reporter

2020 ◽  
pp. jbc.RA120.014553
Author(s):  
Zachary J Kartje ◽  
Helen I Janis ◽  
Shaoni Mukhopadhyay ◽  
Keith T Gagnon
Keyword(s):  
Rna Polymerase ◽  
Real Time ◽  
High Throughput Screening ◽  
In Vitro Transcription ◽  
T7 Rna Polymerase ◽  
Rna Aptamer ◽  
Reaction Conditions ◽  
Fluorescent Reporter ◽  
Transcription In Vitro

Methods for rapid and high-throughput screening of transcription in vitro to examine reaction conditions, enzyme mutants, promoter variants, and small molecule modulators can be extremely valuable tools. However, these techniques may be difficult to establish or inaccessible to many researchers. To develop a straightforward and cost-effective platform for assessing transcription in vitro, we used the “Broccoli” RNA aptamer as a direct, real-time fluorescent transcript readout. To demonstrate the utility of our approach, we screened the effect of common reaction conditions and components on bacteriophage T7 RNA polymerase (RNAP) activity using a common quantitative PCR instrument for fluorescence detection. Several essential conditions for in vitro transcription by T7 RNAP were confirmed with this assay, including the importance of enzyme and substrate concentrations, co-variation of magnesium and nucleoside triphosphates, and the effects of several typical additives. When we used this method to assess all possible point mutants of a canonical T7 RNAP promoter, our results coincided well with previous reports. This approach should translate well to a broad variety of bacteriophage in vitro transcription systems and provides a platform for developing fluorescence-based readouts of more complex transcription systems in vitro.

scholarly journals Evolution of an inhibitory RNA aptamer against T7 RNA polymerase

FEBS Open Bio ◽  
2012 ◽  
Vol 2 (1) ◽  
pp. 203-207 ◽  
Author(s):  
Shoji Ohuchi ◽  
Yusuke Mori ◽  
Yoshikazu Nakamura
Keyword(s):  
Rna Polymerase ◽  
T7 Rna Polymerase ◽  
Rna Aptamer

scholarly journals Stringent control in Escherichia coli applies also to transcription by T7 RNA polymerase.

1987 ◽  
Vol 262 (9) ◽  
pp. 3940-3943
Author(s):  
M. Yamagishi ◽  
J.R. Cole ◽  
M. Nomura ◽  
F.W. Studier ◽  
J.J. Dunn
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
T7 Rna Polymerase ◽  
Stringent Control
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