scholarly journals Rapid and Scalable Preparation of Bacterial Lysates for Cell-Free Gene Expression

2017 ◽  
Vol 6 (12) ◽  
pp. 2198-2208 ◽  
Author(s):  
Andriy Didovyk ◽  
Taishi Tonooka ◽  
Lev Tsimring ◽  
Jeff Hasty
Keyword(s):  
Gene Expression ◽  
Free Gene ◽  
Bacterial Lysates

scholarly journals Rapid and scalable preparation of bacterial lysates for cell-free gene expression

2017 ◽  
Author(s):  
Andriy Didovyk ◽  
Taishi Tonooka ◽  
Lev Tsimring ◽  
Jeff Hasty
Keyword(s):  
Gene Expression ◽  
Synthetic Biology ◽  
Cost Effective ◽  
Diverse Range ◽  
Highly Active ◽  
Freeze Dried ◽  
Wide Range ◽  
Specialized Equipment ◽  
Free Gene ◽  
Bacterial Lysates

AbstractCell-free gene expression systems are emerging as an important platform for a diverse range of synthetic biology and biotechnology applications, including production of robust field-ready biosensors. Here, we combine programmed cellular autolysis with a freeze-thaw or freeze-dry cycle to create a practical, reproducible, and a labor- and cost-effective approach for rapid production of bacterial lysates for cell-free gene expression. Using this method, ro-bust and highly active bacterial cell lysates can be produced without specialized equipment at a wide range of scales, making cell-free gene expression easily and broadly accessible. More-over, live autolysis strain can be freeze-dried directly and subsequently lysed upon rehydration to produce active lysate. We demonstrate the utility of autolysates for synthetic biology by reg-ulating protein production and degradation, implementing quorum sensing, and showing quan-titative protection of linear DNA templates by GamS protein. To allow versatile and sensitive β-galactosidase (LacZ) based readout we produce autolysates with no detectable background LacZ activity and use them to produce sensitive mercury(II) biosensors with LacZ-mediated colorimetric and fluorescent outputs. The autolysis approach can facilitate wider adoption of cell-free technology for cell-free gene expression as well as other synthetic biology and biotechnology applications, such as metabolic engineering, natural product biosynthesis, or proteomics.

scholarly journals Improving cell-free glycoprotein synthesis by characterizing and enriching native membrane vesicles

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jasmine M. Hershewe ◽  
Katherine F. Warfel ◽  
Shaelyn M. Iyer ◽  
Justin A. Peruzzi ◽  
Claretta J. Sullivan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Synthetic Biology ◽  
Membrane Protein ◽  
Membrane Vesicles ◽  
Processing Methods ◽  
Glycoprotein Synthesis ◽  
On Demand ◽  
Free Gene ◽  
Membrane Bound

AbstractCell-free gene expression (CFE) systems from crude cellular extracts have attracted much attention for biomanufacturing and synthetic biology. However, activating membrane-dependent functionality of cell-derived vesicles in bacterial CFE systems has been limited. Here, we address this limitation by characterizing native membrane vesicles in Escherichia coli-based CFE extracts and describing methods to enrich vesicles with heterologous, membrane-bound machinery. As a model, we focus on bacterial glycoengineering. We first use multiple, orthogonal techniques to characterize vesicles and show how extract processing methods can be used to increase concentrations of membrane vesicles in CFE systems. Then, we show that extracts enriched in vesicle number also display enhanced concentrations of heterologous membrane protein cargo. Finally, we apply our methods to enrich membrane-bound oligosaccharyltransferases and lipid-linked oligosaccharides for improving cell-free N-linked and O-linked glycoprotein synthesis. We anticipate that these methods will facilitate on-demand glycoprotein production and enable new CFE systems with membrane-associated activities.

Cell-Free Gene Expression from

2022 ◽  
pp. 135-149
Author(s):  
Michael Levy ◽  
Ohad Vonshak ◽  
Yiftach Divon ◽  
Ferdinand Greiss ◽  
Noa Avidan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Free Gene

Branched DNA Architectures Produced by PCR‐Based Assembly as Gene Compartments for Cell‐Free Gene‐Expression Reactions

ChemBioChem ◽  
2019 ◽  
Vol 20 (20) ◽  
pp. 2597-2603 ◽  
Author(s):  
Xiaocui Guo ◽  
Lihui Bai ◽  
Feng Li ◽  
Wilhelm T. S. Huck ◽  
Dayong Yang
Keyword(s):  
Gene Expression ◽  
Branched Dna ◽  
Free Gene

Dye-Free Gene Expression Detection by Sequence-Tagged Reverse-Transcription Polymerase Chain Reaction Coupled with Pyrosequencing

2009 ◽  
Vol 81 (1) ◽  
pp. 273-281 ◽  
Author(s):  
Xiaodan Zhang ◽  
Haiping Wu ◽  
Zhiyao Chen ◽  
Guohua Zhou ◽  
Tomoharu Kajiyama ◽  
...  
Keyword(s):  
Gene Expression ◽  
Polymerase Chain Reaction ◽  
Reverse Transcription ◽  
Chain Reaction ◽  
Free Gene ◽  
Polymerase Chain

Cover Picture: A Single-Step Photolithographic Interface for Cell-Free Gene Expression and Active Biochips (Small 3/2007)

Small ◽  
2007 ◽  
Vol 3 (3) ◽  
pp. 349-349
Author(s):  
Amnon Buxboim ◽  
Maya Bar-Dagan ◽  
Veronica Frydman ◽  
David Zbaida ◽  
Margherita Morpurgo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Step ◽  
Free Gene

scholarly journals Reconstruction of phospholipid synthesis by combing in vitro fatty acid synthesis and cell-free gene expression

2021 ◽  
Author(s):  
Sumie Eto ◽  
Rumie Matsumura ◽  
Mai Fujimi ◽  
Yasuhiro Shimane ◽  
Samuel Berhanu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Fatty Acid ◽  
Fatty Acid Synthesis ◽  
Acid Synthesis ◽  
Phospholipid Synthesis ◽  
Giant Vesicles ◽  
Phosphatidic Acids ◽  
A Cell ◽  
Free Gene

Phospholipid synthesis is a fundamental process that promotes cell propagation and, presently, is the most challenging issue in artificial cell research aimed at reconstituting living cells from biomolecules. Here, we constructed a cell-free phospholipid synthesis system that combines in vitro fatty acid synthesis and a cell-free gene expression system that synthesizes acyltransferases for phospholipid synthesis. Fatty acids were synthesized from acetyl-CoA and malonyl-CoA, then continuously converted into phosphatidic acids by the cell-free synthesized acyltransferases. Because the system can avoid the accumulation of synthetic intermediates that suppress the reaction, the yield of phospholipid has significantly improved from previous schemes (up to 400 μM). Additionally, by adding enzymes for recycling CoA, we synthesized phosphatidic acids from acetic acid and bicarbonate as carbon sources. The constructed system is available to express the genes from pathogenic bacteria and to analyze the synthesized phospholipids. By encapsulating our system inside giant vesicles, it would be possible to construct the artificial cells in which the membrane grows and divides sustainably.

scholarly journals Cell-free gene expression

2021 ◽  
Vol 1 (1) ◽  
Author(s):  
David Garenne ◽  
Matthew C. Haines ◽  
Eugenia F. Romantseva ◽  
Paul Freemont ◽  
Elizabeth A. Strychalski ◽  
...  
Keyword(s):  
Gene Expression ◽  
Free Gene
Sign in / Sign up

Export Citation Format

Share Document