Protecting Linear DNA Templates in Cell-Free Expression Systems from Diverse Bacteria

2020 ◽  
Vol 9 (10) ◽  
pp. 2851-2855
Author(s):  
Sung Sun Yim ◽  
Nathan I. Johns ◽  
Vincent Noireaux ◽  
Harris H. Wang
Keyword(s):  
Free Expression ◽  
Expression Systems ◽  
Dna Templates ◽  
Linear Dna

scholarly journals Akaby - cell-free protein expression system for linear templates

2021 ◽  
Author(s):  
Wakana Sato ◽  
Judee Sharon ◽  
Christopher Deich ◽  
Nathaniel Gaut ◽  
Brock Cash ◽  
...  
Keyword(s):  
Protein Expression ◽  
Expression System ◽  
Nuclease Activity ◽  
Coli Strain ◽  
Free Expression ◽  
Dna Templates ◽  
Free Protein ◽  
E Coli ◽  
Linear Dna ◽  
Simple Alternative

Cell-free protein expression is increasingly becoming popular for biotechnology, biomedical and research applications. Among cell-free systems, the most popular one is based on Escherichia coli (E. coli). Endogenous nucleases in E. coli cell-free transcription-translation (TXTL) degrade the free ends of DNA, resulting in inefficient protein expression from linear DNA templates. RecBCD is a nuclease complex that plays a major role in nuclease activity in E. coli, with the RecB subunit possessing the actual nuclease activity. We created a RecB knockout of an E. coli strain optimized for cell-free expression. We named this new strain Akaby. We demonstrated that Akaby TXTL successfully reduced linear DNA degradations, rescuing the protein expression efficiency from the linear DNA templates. The practicality of Akaby for TXTL is an efficient, simple alternative for linear template expression in cell-free reactions. We also use this work as a model protocol for modifying the TXTL source E. coli strain, enabling the creation of TXTL systems with other custom modifications.

scholarly journals Effective Use of Linear DNA in Cell-Free Expression Systems

2021 ◽  
Vol 9 ◽  
Author(s):  
Megan A. McSweeney ◽  
Mark P. Styczynski
Keyword(s):  
Scientific Discovery ◽  
Free Expression ◽  
Genetic Circuit ◽  
Expression Systems ◽  
Linear Dna ◽  
Biological Phenomena ◽  
Wide Range ◽  
Research Tools

Cell-free expression systems (CFEs) are cutting-edge research tools used in the investigation of biological phenomena and the engineering of novel biotechnologies. While CFEs have many benefits over in vivo protein synthesis, one particularly significant advantage is that CFEs allow for gene expression from both plasmid DNA and linear expression templates (LETs). This is an important and impactful advantage because functional LETs can be efficiently synthesized in vitro in a few hours without transformation and cloning, thus expediting genetic circuit prototyping and allowing expression of toxic genes that would be difficult to clone through standard approaches. However, native nucleases present in the crude bacterial lysate (the basis for the most affordable form of CFEs) quickly degrade LETs and limit expression yield. Motivated by the significant benefits of using LETs in lieu of plasmid templates, numerous methods to enhance their stability in lysate-based CFEs have been developed. This review describes approaches to LET stabilization used in CFEs, summarizes the advancements that have come from using LETs with these methods, and identifies future applications and development goals that are likely to be impactful to the field. Collectively, continued improvement of LET-based expression and other linear DNA tools in CFEs will help drive scientific discovery and enable a wide range of applications, from diagnostics to synthetic biology research tools.

scholarly journals Local deuteration enables NMR observation of methyl groups in proteins from eukaryotic and cell‐free expression systems.

2021 ◽  
Author(s):  
Abhinav Dubey ◽  
Nikolay Stoyanov ◽  
Thibault Viennet ◽  
Sandeep Chhabra ◽  
Shantha Elter ◽  
...  
Keyword(s):  
Free Expression ◽  
Methyl Groups ◽  
Expression Systems

Co-translational Stabilization of Insoluble Proteins in Cell-Free Expression Systems

2014 ◽  
pp. 125-143 ◽  
Author(s):  
Lei Kai ◽  
Erika Orbán ◽  
Erik Henrich ◽  
Davide Proverbio ◽  
Volker Dötsch ◽  
...  
Keyword(s):  
Free Expression ◽  
Expression Systems

scholarly journals Data on multimerization efficiency for short linear DNA templates and phosphoryl guanidine primers during isothermal amplification with Bst exo- DNA polymerase

Data in Brief ◽  
2020 ◽  
Vol 29 ◽  
pp. 105188 ◽  
Author(s):  
Ravil R. Garafutdinov ◽  
Assol R. Sakhabutdinova ◽  
Maxim S. Kupryushkin ◽  
Dmitrii V. Pyshnyi
Keyword(s):  
Dna Polymerase ◽  
Isothermal Amplification ◽  
Dna Templates ◽  
Linear Dna

scholarly journals Cell-Free Synthetic Biology Platform for Engineering Synthetic Biological Circuits and Systems

2019 ◽  
Vol 2 (2) ◽  
pp. 39 ◽  
Author(s):  
Dohyun Jeong ◽  
Melissa Klocke ◽  
Siddharth Agarwal ◽  
Jeongwon Kim ◽  
Seungdo Choi ◽  
...  
Keyword(s):  
Synthetic Biology ◽  
Free Expression ◽  
Dna Nanostructures ◽  
Expression Systems ◽  
Natural Systems ◽  
The Past ◽  
Current Cell ◽  
Recent Developments ◽  
Biological Circuits

Synthetic biology integrates diverse engineering disciplines to create novel biological systems for biomedical and technological applications. The substantial growth of the synthetic biology field in the past decade is poised to transform biotechnology and medicine. To streamline design processes and facilitate debugging of complex synthetic circuits, cell-free synthetic biology approaches has reached broad research communities both in academia and industry. By recapitulating gene expression systems in vitro, cell-free expression systems offer flexibility to explore beyond the confines of living cells and allow networking of synthetic and natural systems. Here, we review the capabilities of the current cell-free platforms, focusing on nucleic acid-based molecular programs and circuit construction. We survey the recent developments including cell-free transcription–translation platforms, DNA nanostructures and circuits, and novel classes of riboregulators. The links to mathematical models and the prospects of cell-free synthetic biology platforms will also be discussed.

Assembling Linear DNA Templates for In Vitro Transcription and Translation

2011 ◽  
pp. 67-78
Author(s):  
Viktor Stein ◽  
Miriam Kaltenbach ◽  
Florian Hollfelder
Keyword(s):  
In Vitro Transcription ◽  
Dna Templates ◽  
Linear Dna

scholarly journals Metabolic Profiling of Escherichia coli-Based Cell-Free Expression Systems for Process Optimization

2019 ◽  
Vol 58 (50) ◽  
pp. 22472-22482 ◽  
Author(s):  
April M. Miguez ◽  
Monica P. McNerney ◽  
Mark P. Styczynski
Keyword(s):  
Escherichia Coli ◽  
Process Optimization ◽  
Metabolic Profiling ◽  
Free Expression ◽  
Expression Systems

scholarly journals Functional Expression of the PorAH Channel fromCorynebacterium glutamicumin Cell-free Expression Systems

2011 ◽  
Vol 286 (37) ◽  
pp. 32525-32532 ◽  
Author(s):  
Parthasarathi Rath ◽  
Pascal Demange ◽  
Olivier Saurel ◽  
Marielle Tropis ◽  
Mamadou Daffé ◽  
...  
Keyword(s):  
Functional Expression ◽  
Free Expression ◽  
Expression Systems
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