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

A Single-Step Photolithographic Interface for Cell-Free Gene Expression and Active Biochips

Small ◽  
2007 ◽  
Vol 3 (3) ◽  
pp. 500-510 ◽  
Author(s):  
Amnon Buxboim ◽  
Maya Bar-Dagan ◽  
Veronica Frydman ◽  
David Zbaida ◽  
Margherita Morpurgo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Step ◽  
Free Gene

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

scholarly journals Production of Recombinant and Tagged Proteins in the Hyperthermophilic Archaeon Sulfolobus solfataricus

2006 ◽  
Vol 72 (1) ◽  
pp. 102-111 ◽  
Author(s):  
S.-V. Albers ◽  
M. Jonuscheit ◽  
S. Dinkelaker ◽  
T. Urich ◽  
A. Kletzin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Shuttle Vector ◽  
Sulfolobus Solfataricus ◽  
Inducible Promoter ◽  
Single Step ◽  
Vector System ◽  
Hyperthermophilic Archaea ◽  
Model Organisms ◽  
Hyperthermophilic Archaeon ◽  
High Level

ABSTRACT Many systems are available for the production of recombinant proteins in bacterial and eukaryotic model organisms, which allow us to study proteins in their native hosts and to identify protein-protein interaction partners. In contrast, only a few transformation systems have been developed for archaea, and no system for high-level gene expression existed for hyperthermophilic organisms. Recently, a virus-based shuttle vector with a reporter gene was developed for the crenarchaeote Sulfolobus solfataricus, a model organism of hyperthermophilic archaea that grows optimally at 80°C (M. Jonuscheit, E. Martusewitsch, K. M. Stedman, and C. Schleper, Mol. Microbiol. 48:1241-1252, 2003). Here we have refined this system for high-level gene expression in S. solfataricus with the help of two different promoters, the heat-inducible promoter of the major chaperonin, thermophilic factor 55, and the arabinose-inducible promoter of the arabinose-binding protein AraS. Functional expression of heterologous and homologous genes was demonstrated, including production of the cytoplasmic sulfur oxygenase reductase from Acidianus ambivalens, an Fe-S protein of the ABC class from S. solfataricus, and two membrane-associated ATPases potentially involved in the secretion of proteins. Single-step purification of the proteins was obtained via fused His or Strep tags. To our knowledge, these are the first examples of the application of an expression vector system to produce large amounts of recombinant and also tagged proteins in a hyperthermophilic archaeon.

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

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
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