scholarly journals Self-Organization Controls Expression More than Abundance of Molecular Components of Transcription and Translation in Confined Cell-Free Gene Expression

2018 ◽  
Author(s):  
Patrick M. Caveney ◽  
Rosemary M. Dabbs ◽  
Gaurav Chauhan ◽  
S. Elizabeth Norred ◽  
C. Patrick Collier ◽  
...  
Keyword(s):  
Gene Expression ◽  
Self Organization ◽  
Free Gene ◽  
Molecular Components

scholarly journals Self-organization controls expression more than abundance of molecular components of transcription and translation in confined cell-free gene expression

2018 ◽  
Author(s):  
P.M. Caveney ◽  
R. Dabbs ◽  
G. Chauhan ◽  
S.E. Norred ◽  
C.P. Collier ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Production ◽  
Self Organization ◽  
Large Variability ◽  
Expression Noise ◽  
Expression Efficiency ◽  
Cell Extracts ◽  
Reaction Chambers ◽  
Free Gene ◽  
Molecular Components

AbstractCell-free gene expression using purified components or cell extracts has become an important platform for synthetic biology that is finding a growing numBer of practical applications. Unfortunately, at cell-relevant reactor volumes, cell-free expression suffers from excessive variability (noise) such that protein concentrations may vary by more than an order of magnitude across a population of identically constructed reaction chambers. Consensus opinion holds that variability in expression is due to the stochastic distribution of expression resources (DNA, RNAP, ribosomes, etc.) across the population of reaction chambers. In contrast, here we find that chamber-to-chamber variation in the expression efficiency generates the large variability in protein production. Through analysis and modeling, we show that chambers self-organize into expression centers that control expression efficiency. Chambers that organize into many centers, each having relatively few expression resources, exhibit high expression efficiency. Conversely, chambers that organize into just a few centers where each center has an abundance of resources, exhibit low expression efficiency. A particularly surprising finding is that diluting expression resources reduces the chamber-to-chamber variation in protein production. Chambers with dilute pools of expression resources exhibit higher expression efficiency and lower expression noise than those with more concentrated expression resources. In addition to demonstrating the means to tune expression noise, these results demonstrate that in cell-free systems, self-organization may exert even more influence over expression than the abundance of the molecular components of transcription and translation. These observations in cell-free platform may elucidate how self-organized, membrane-less structures emerge and function in cells.

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
Sign in / Sign up

Export Citation Format

Share Document