scholarly journals A method for cost-effective and rapid characterization of engineered T7-based transcription factors by cell-free protein synthesis reveals insights into the regulation of T7 RNA polymerase-driven expression

2019 ◽  
Vol 674 ◽  
pp. 108045 ◽  
Author(s):  
John B. McManus ◽  
Peter A. Emanuel ◽  
Richard M. Murray ◽  
Matthew W. Lux
Keyword(s):  
Protein Synthesis ◽  
Transcription Factors ◽  
Rna Polymerase ◽  
Cost Effective ◽  
T7 Rna Polymerase ◽  
Free Protein ◽  
Rapid Characterization ◽  
Cell Free Protein Synthesis

scholarly journals A Method for Cost-Effective and Rapid Characterization of Engineered T7-based Transcription Factors by Cell-Free Protein Synthesis Reveals Insights into the Regulation of T7 RNA Polymerase-Driven Expression

2019 ◽  
Author(s):  
John B. McManus ◽  
Richard M. Murray ◽  
Peter A. Emanuel ◽  
Matthew W. Lux
Keyword(s):  
Protein Synthesis ◽  
Transcription Factors ◽  
Protein Expression ◽  
Rna Polymerase ◽  
Circuit Design ◽  
Cost Effective ◽  
Gene Circuit ◽  
Free Protein ◽  
Cost Effective Method ◽  
Cell Free Protein Synthesis

AbstractThe T7 bacteriophage RNA polymerase (T7 RNAP) serves as a model for understanding RNA synthesis, as a tool for protein expression, and as an actuator for synthetic gene circuit design in bacterial cells and cell-free extract. T7 RNAP is an attractive tool for orthogonal protein expression in bacteria owing to its compact single subunit structure and orthogonal promoter specificity. Understanding the mechanisms underlying T7 RNAP regulation is important to the design of engineered T7-based transcription factors, which can be used in gene circuit design. To explore regulatory mechanisms for T7 RNAP-driven expression, we developed a rapid and cost-effective method to characterize engineered T7-based transcription factors using cell-free protein synthesis and an acoustic liquid handler. Using this method, we investigated the effects of the tetracycline operator’s proximity to the T7 promoter on the regulation of T7 RNAP-driven expression. Our results reveal a mechanism for regulation that functions by interfering with the transition of T7 RNAP from initiation to elongation and validates the use of the method described here to engineer future T7-based transcription factors.HighlightsDevelopment of a rapid and cost-effective method for screening synthetic promoters.Insights into the regulation of engineered T7-based transcription factors and T7 RNAP enzyme kinetics.Validation of this method by comparison with the T7 RNAP kinetic model.

scholarly journals Rapid production and characterization of antimicrobial colicins using Escherichia coli-based cell-free protein synthesis

2018 ◽  
Vol 3 (1) ◽  
Author(s):  
Xing Jin ◽  
Weston Kightlinger ◽  
Yong-Chan Kwon ◽  
Seok Hoon Hong
Keyword(s):  
Escherichia Coli ◽  
Protein Synthesis ◽  
Host Cells ◽  
Persister Cells ◽  
Free Protein ◽  
E Coli ◽  
Dna And Rna ◽  
Rapid Production ◽  
Cell Free Protein Synthesis

Abstract Colicins are antimicrobial proteins produced by Escherichia coli, which, upon secretion from the host, kill non-host E. coli strains by forming pores in the inner membrane and degrading internal cellular components such as DNA and RNA. Due to their unique cell-killing activities, colicins are considered viable alternatives to conventional antibiotics. Recombinant production of colicins requires co-production of immunity proteins to protect host cells; otherwise, the colicins are lethal to the host. In this study, we used cell-free protein synthesis (CFPS) to produce active colicins without the need for protein purification and co-production of immunity proteins. Cell-free synthesized colicins were active in killing model E. coli cells with different modes of cytotoxicity. Pore-forming colicins E1 and nuclease colicin E2 killed actively growing cells in a nutrient-rich medium, but the cytotoxicity of colicin Ia was low compared to E1 and E2. Moreover, colicin E1 effectively killed cells in a nutrient-free solution, while the activity of E2 was decreased compared to nutrient-rich conditions. Both colicins E1 and E2 decreased the level of persister cells (metabolically dormant cell populations that are insensitive to antibiotics) by up to six orders of magnitude compared to that of the rifampin pretreated persister cells. This study finds that colicins can eradicate non-growing cells including persisters, and that CFPS is a promising platform for rapid production and characterization of toxic proteins.

scholarly journals Preparation and partial characterization of cell-free protein-synthesizing extracts from Tetrahymena pyriformis

1981 ◽  
Vol 194 (3) ◽  
pp. 761-770 ◽  
Author(s):  
E T David ◽  
K E Smith
Keyword(s):  
Protein Synthesis ◽  
Tetrahymena Pyriformis ◽  
Partial Characterization ◽  
Free Protein ◽  
Optimum Synthesis ◽  
Kinetics Of ◽  
Cell Free Protein Synthesis ◽  
Kinetics Of Protein Synthesis

1. We have examined methods necessary for preparing post-mitochondrial supernatants from Tetrahymena pyriformis strain HSM that are capable of efficient cell-free protein synthesis. 2. The requirements for optimum synthesis in these extracts are described. 3. Data relating to the kinetics of protein synthesis and the initiation capacity of these supernatants are presented.

Simple procedures for the construction of a robust and cost-effective cell-free protein synthesis system

2006 ◽  
Vol 126 (4) ◽  
pp. 554-561 ◽  
Author(s):  
Tae-Wan Kim ◽  
Jung-Won Keum ◽  
In-Seok Oh ◽  
Cha-Yong Choi ◽  
Chang-Gil Park ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Cost Effective ◽  
Synthesis System ◽  
Free Protein ◽  
Protein Synthesis System ◽  
Cell Free Protein Synthesis

scholarly journals Isolation and Characterization of a Novel Perchloric Acid-soluble Protein Inhibiting Cell-free Protein Synthesis

1995 ◽  
Vol 270 (50) ◽  
pp. 30060-30067 ◽  
Author(s):  
Tatsuzo Oka ◽  
Hideaki Tsuji ◽  
Chie Noda ◽  
Kentaro Sakai ◽  
Yeong-man Hong ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Perchloric Acid ◽  
Soluble Protein ◽  
Free Protein ◽  
Isolation And Characterization ◽  
Cell Free Protein Synthesis
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