scholarly journals Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System

10.3791/56144 ◽  
2017 ◽  
Author(s):  
Mark Rustad ◽  
Allen Eastlund ◽  
Ryan Marshall ◽  
Paul Jardine ◽  
Vincent Noireaux
Keyword(s):  
Expression System ◽  
Free Expression ◽  
E Coli ◽  
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>

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 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 Optimization of ClpXP activity and protein synthesis in an E. coli extract-based cell-free expression system

2018 ◽  
Vol 8 (1) ◽  
Author(s):  
Xinying Shi ◽  
Ti Wu ◽  
Christian M. Cole ◽  
Neal K. Devaraj ◽  
Simpson Joseph
Keyword(s):  
Protein Synthesis ◽  
Expression System ◽  
Free Expression ◽  
E Coli ◽  
Cell Free Expression System

Mechanism of estrogen receptor-dependent transcription in a cell-free system

1990 ◽  
Vol 10 (12) ◽  
pp. 6607-6612
Author(s):  
J F Elliston ◽  
S E Fawell ◽  
L Klein-Hitpass ◽  
S Y Tsai ◽  
M J Tsai ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
Rna Polymerase Ii ◽  
Expression System ◽  
Steroid Receptor ◽  
Free Expression ◽  
Independent Manner ◽  
Free System ◽  
A Cell ◽  
Regulated Gene Expression ◽  
Cell Free Expression System

RNA synthesis was stimulated directly in a cell-free expression system by crude preparations of recombinant mouse estrogen receptor (ER). Receptor-stimulated transcription required the presence of estrogen response elements (EREs) in the test template and could be specifically inhibited by addition of competitor oligonucleotides containing EREs. Moreover, polyclonal antibodies directed against the DNA-binding region of ER inhibited ER-dependent transcription. In our cell-free expression system, hormone-free ER induced transcription in a hormone-independent manner. Evidence is presented suggesting that ER acts by facilitating the formation of a stable preinitiation complex at the target gene promoter and thus augments the initiation of transcription by RNA polymerase II. These observations lend support to our current understanding of the mechanism of steroid receptor-regulated gene expression and suggest strong conservation of function among members of the steroid receptor superfamily.

Abstract 437: Optimizing a Prokaryotic Cell Free Expression System for the Generation of synthetic ApoB-Containing Lipoprotein Particles

2013 ◽  
Vol 33 (suppl_1) ◽  
Author(s):  
Humra Athar ◽  
Zhenghui G Jiang ◽  
Christopher J McKnight
Keyword(s):  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Expression System ◽  
Low Density Lipoproteins ◽  
Free Expression ◽  
Low Density ◽  
Prokaryotic Cell ◽  
Lipoprotein Particles ◽  
Cell Free Expression System

High serum levels of low density lipoproteins (LDL) is associated with increased risk of atherosclerosis. Apolipoprotein B (ApoB) is required for the assembly and secretion of chylomicrons and very low density lipoproteins (VLDL), the precursor of low density lipoproteins (LDL). Despite its clinical significance, the mechanism of the assembly of these ApoB containing lipoproteins is poorly understood. The assembly process is an interplay of several key components including but not limited to nascent ApoB, lipids, ER resident chaperones and importantly, microsomal triglyceride transfer protein (MTP). In the current study, we are trying to understand several unanswered questions in the mechanism of the lipoprotein assembly. We have used a novel prokaryotic cell-free expression system and lipids mimicking the ER membrane to produce particles that represent the early dense initiation particles formed in the ER. After optimizing several different conditions, we were able to make “synthetic” lipoproteins by cotranslational expression of constructs from the first 22% of ApoB tagged with a 6-histidine tag at the C-terminus (ApoB 22-His) with small unilamellar phosphatidylcholine (PC) vesicles and phosphatidylcholine:triolein (PC:TO) emulsions. After cotranslational interaction with lipids, these constructs migrate to a lower density in potassium bromide (KBr) density gradient centrifugation. Here we report a new ApoB 22 construct with a FLAG tag at the N-terminus in addition to the C-terminal His tag. The construct makes significant amount of soluble protein that is soluble in the cell free reaction. The two N- and C-terminal tags allow us to purify full length construct from any truncation products. In addition, the dual-tag approach will allow us to purify the synthetic lipoproteins directly from the cell free system, and thereby avoid the requirement for KBr density gradient centrifugation. This new strategy will provide far more efficient generation and purification of synthetic ApoB containing lipoprotein particles.

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