scholarly journals Protein degradation in a TX-TL cell-free expression system using ClpXP protease

2015 ◽  
Author(s):  
Zachary Sun ◽  
Jongmin Kim ◽  
Vipul Singhal ◽  
Richard M Murray
Keyword(s):  
Protein Degradation ◽  
Expression System ◽  
Free Expression ◽  
Batch Mode ◽  
Protein Targets ◽  
Resource Limited ◽  
Escherichia Coli Expression ◽  
Cell Free Expression System

An in vitro S30-based Escherichia coli expression system (“Transcription-Translation”, or “TX-TL”) has been developed as an alternative prototyping environment to the cell for synthetic circuits [1-5]. Basic circuit elements, such as switches and cascades, have been shown to function in TX-TL, as well as bacteriophage assembly [2, 6]. Circuits can also be prototyped from basic parts within 8 hours, avoiding cloning and transformation steps [7]. However, most published results have been obtained in a “batch mode” reaction, where factors that play an important role for in vivo circuit dynamics – namely protein degradation and protein dilution – are severely hindered or are not present. This limits the complexity of circuits built in TX-TL without steady-state or continuous-flow solutions [8-10]. However, alternate methods that enable dilution either require extra equipment and expertise or demand lower reaction throughput. We explored the possibility of supplementing TX-TL with ClpXP, an AAA+ protease pair that selectively degrades tagged proteins [11], to provide finely-tuned degradation. The mechanism of ClpXP degradation has been extensively studied both in vitro and in vivo [12-15]. However, it has not been characterized for use in synthetic circuits – metrics such as toxicity, ATP usage, degradation variation over time, and cellular loading need to be determined. In particular, TX-TL in batch mode is known to be resource limited [16], and ClpXP is known to require significant amounts of ATP to unfold different protein targets [17, 18]. We find that ClpXP’s protein degradation dynamics is dependent on protein identity, but can be determined experimentally. Degradation follows Michaels-Menten kinetics, and can be fine tuned by ClpX or ClpP concentration. Added purified ClpX is also not toxic to TX-TL reactions. Therefore, ClpXP provides a controllable way to introduce protein degradation and dynamics into synthetic circuits in TX-TL.

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 Incompatibility of DFHBI based fluorescent RNA aptamers with particular commercial cell-free expression systems

2021 ◽  
Author(s):  
Alexander J Speakman ◽  
Katherine E Dunn
Keyword(s):  
Expression System ◽  
In Vitro Transcription ◽  
Rna Aptamers ◽  
Free Expression ◽  
Reaction Products ◽  
Expression Systems ◽  
E Coli ◽  
Cell Free Expression System

Fluorescent RNA aptamers are an increasingly used tool for quantifying transcription and for visualising RNA interactions, both in vitro and in vivo. However when tested in the commercially available, E. coli extract based Expressway™ cell-free expression system, no fluorescence is detected. The same experimental setup is shown to successfully produce fluorescent RNA aptamers when tested in another buffer designed for in vitro transcription, and RNA purification of the Expressway™ reaction products show that transcription does occur, but does not result in a fluorescent product. In this paper we demonstrate the incompatibility of a narrow selection of RNA aptamers in one particular cell-free expression system, and consider that similar issues may arise with other cell-free expression systems, RNA aptamers, and their corresponding fluorophores.

scholarly journals Efficacy of a Potential Trivalent Vaccine Based on Hc Fragments of Botulinum Toxins A, B, and E Produced in a Cell-Free Expression System

2010 ◽  
Vol 17 (5) ◽  
pp. 784-792 ◽  
Author(s):  
R. Zichel ◽  
A. Mimran ◽  
A. Keren ◽  
A. Barnea ◽  
I. Steinberger-Levy ◽  
...  
Keyword(s):  
Expression System ◽  
Free Expression ◽  
Enzyme Linked Immunosorbent Assay ◽  
High Dose ◽  
Botulinum Toxins ◽  
Free System ◽  
Toxin Complex ◽  
A Cell ◽  
Cell Free Expression System

ABSTRACT Botulinum toxins produced by the anaerobic bacterium Clostridium botulinum are the most potent biological toxins in nature. Traditionally, people at risk are immunized with a formaldehyde-inactivated toxin complex. Second generation vaccines are based on the recombinant carboxy-terminal heavy-chain (Hc) fragment of the neurotoxin. However, the materialization of this approach is challenging, mainly due to the high AT content of clostridial genes. Herein, we present an alternative strategy in which the native genes encoding Hc proteins of botulinum toxins A, B, and E were used to express the recombinant Hc fragments in a cell-free expression system. We used the unique property of this open system to introduce different combinations of chaperone systems, protein disulfide isomerase (PDI), and reducing/oxidizing environments directly to the expression reaction. Optimized expression conditions led to increased production of soluble Hc protein, which was successfully scaled up using a continuous exchange (CE) cell-free system. Hc proteins were produced at a concentration of more than 1 mg/ml and purified by one-step Ni+ affinity chromatography. Mice immunized with three injections containing 5 μg of any of the in vitro-expressed, alum-absorbed, Hc vaccines generated a serum enzyme-linked immunosorbent assay (ELISA) titer of 105 against the native toxin complex, which enabled protection against a high-dose toxin challenge (103 to 106 mouse 50% lethal dose [MsLD50]). Finally, immunization with a trivalent HcA, HcB, and HcE vaccine protected mice against the corresponding trivalent 105 MsLD50 toxin challenge. Our results together with the latest developments in scalability of the in vitro protein expression systems offer alternative routes for the preparation of botulinum vaccine.

scholarly journals A Methylation-Directed, Synthetic Pap Switch Based on Self-Complementary Regulatory DNA in an All-E. Coli Cell-Free Expression System

2020 ◽  
Author(s):  
Emanuel Worst ◽  
oemer Kurt ◽  
Marc Finkler ◽  
Marc Schenkelberger ◽  
Vincent Noireaux ◽  
...  
Keyword(s):  
Expression System ◽  
Regulatory Region ◽  
Phase Variation ◽  
Coli Strain ◽  
Coli Cell ◽  
Free Expression ◽  
E Coli ◽  
Regulatory Dna ◽  
Cell Free Expression System

<p>Pyelonephritis-associated pili (pap) enable migration of the uropathogenic Escherichia coli strain (UPEC) through the urinary tract. UPEC can switch between a stable 'ON phase' where the corresponding pap genes are expressed and a stable 'OFF phase' where their transcription is repressed. Hereditary, alternate DNA methylation of only two GATC motives within the regulatory region stabilizes the respective phase over many generations. The underlying molecular mechanism is only partly understood. Previous investigations suggest that in vivo phase-variation stability results from cooperative action of the transcriptional regulators Lrp and PapI. Here, we use an E. coli cell-free expression system to study the function of pap regulatory region based on a specially designed, synthetic construct flanked by two reporter genes encoding fluorescent proteins for simple readout. Based on our observations we suggest that Lrp and the conformation of the self-complementary regulatory DNA play a strong role in the regulation of phase-variation. Our work not only contributes to better understand the phase variation mechanism, but it represents a successful start for engineering stable, hereditary and strong expression control based on methylation.</p>

scholarly journals In vitro reconstituted quorum sensing pathways enable rapid evaluation of quorum sensing inhibitors

2021 ◽  
Author(s):  
Dingchen Yu
Keyword(s):  
Quorum Sensing ◽  
Drug Screening ◽  
Expression System ◽  
Communication Process ◽  
Free Expression ◽  
Screening Methods ◽  
High Throughput Drug Screening ◽  
Quorum Sensing Inhibitors

Quorum sensing, as inner- or inter-species microbial communication process orchestrated by diffusible autoinducers, typically results in collective pathogenic behaviours, being recognized as a promising druggable target for anti-virulence. Here, we reconstituted las and rhl quorum sensing pathways of Pseudomonas aeruginosa, mediated by acyl-homoserine lactones (AHLs) and LuxI/LuxR-family proteins, with fluorescence output in Escherichia coli cell-free expression system, offering a platform to rapidly evaluate quorum sensing inhibitors (QSIs) in vitro. Previously reported small-molecule quorum sensing inhibitors for interfering with P. aeruginosa quorum sensing systems were tested and showed mild to high on-target inhibition as well as off-target toxicity. Of note, quercetin displayed potent on-target inhibition to quorum sensing pathways as well as acceptable off-target toxicity to cell-free expression machinery. Upon our work, cell-free platform is anticipated to further facilitate automated and high-throughput drug screening, bridge in silico and in vivo drug-screening methods, and accelerate the upgrading of antimicrobial arsenal.

scholarly journals Cell-free expression of RNA encoded genes using MS2 replicase

2019 ◽  
Vol 47 (20) ◽  
pp. 10956-10967 ◽  
Author(s):  
Laura I Weise ◽  
Michael Heymann ◽  
Viktoria Mayr ◽  
Hannes Mutschler
Keyword(s):  
Gene Expression ◽  
Expression System ◽  
Free Expression ◽  
Genetic Circuits ◽  
Dna And Rna ◽  
Reverse Complement ◽  
Recombinant Cell ◽  
Cell Free Expression System ◽  
Transcription And Replication

AbstractRNA replicases catalyse transcription and replication of viral RNA genomes. Of particular interest for in vitro studies are phage replicases due to their small number of host factors required for activity and their ability to initiate replication in the absence of any primers. However, the requirements for template recognition by most phage replicases are still only poorly understood. Here, we show that the active replicase of the archetypical RNA phage MS2 can be produced in a recombinant cell-free expression system. We find that the 3′ terminal fusion of antisense RNAs with a domain derived from the reverse complement of the wild type MS2 genome generates efficient templates for transcription by the MS2 replicase. The new system enables DNA-independent gene expression both in batch reactions and in microcompartments. Finally, we demonstrate that MS2-based RNA-dependent transcription-translation reactions can be used to control DNA-dependent gene expression by encoding a viral DNA-dependent RNA polymerase on a MS2 RNA template. Our study sheds light on the template requirements of the MS2 replicase and paves the way for new in vitro applications including the design of genetic circuits combining both DNA- and RNA-encoded systems.

In vitro human cell-free expression system for synthesis of malaria proteins

2012 ◽  
Vol 111 (6) ◽  
pp. 2461-2465
Author(s):  
R. Yadavalli ◽  
C. Ledger ◽  
T. Y. Sam-Yellowe
Keyword(s):  
Human Cell ◽  
Expression System ◽  
Free Expression ◽  
Cell Free Expression System

scholarly journals A Methylation-Directed, Synthetic Pap Switch Based on Self-Complementary Regulatory DNA in an All-E. Coli Cell-Free Expression System

2020 ◽  
Author(s):  
Emanuel Worst ◽  
oemer Kurt ◽  
Marc Finkler ◽  
Marc Schenkelberger ◽  
Vincent Noireaux ◽  
...  
Keyword(s):  
Expression System ◽  
Regulatory Region ◽  
Phase Variation ◽  
Coli Strain ◽  
Coli Cell ◽  
Free Expression ◽  
E Coli ◽  
Regulatory Dna ◽  
Cell Free Expression System

<p>Pyelonephritis-associated pili (pap) enable migration of the uropathogenic Escherichia coli strain (UPEC) through the urinary tract. UPEC can switch between a stable 'ON phase' where the corresponding pap genes are expressed and a stable 'OFF phase' where their transcription is repressed. Hereditary, alternate DNA methylation of only two GATC motives within the regulatory region stabilizes the respective phase over many generations. The underlying molecular mechanism is only partly understood. Previous investigations suggest that in vivo phase-variation stability results from cooperative action of the transcriptional regulators Lrp and PapI. Here, we use an E. coli cell-free expression system to study the function of pap regulatory region based on a specially designed, synthetic construct flanked by two reporter genes encoding fluorescent proteins for simple readout. Based on our observations we suggest that Lrp and the conformation of the self-complementary regulatory DNA play a strong role in the regulation of phase-variation. Our work not only contributes to better understand the phase variation mechanism, but it represents a successful start for engineering stable, hereditary and strong expression control based on methylation.</p>

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