scholarly journals Quantifying cellular capacity to identify gene expression designs with reduced burden

2014 ◽  
Author(s):  
Francesca Ceroni ◽  
Rhys J R Algar ◽  
Guy-Bart Stan ◽  
Tom Ellis
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Heterologous Gene Expression ◽  
Heterologous Gene ◽  
E Coli ◽  
Equivalent Expression ◽  
Significant Burden ◽  
Cellular Capacity ◽  
Identify Gene Expression

Heterologous gene expression can be a significant burden to cells, consuming resources and causing decreased growth and stability. We describe here anin vivomonitor that tracksE. colicapacity changes in real-time and can be used to assay the burden synthetic constructs and their parts impose. By measuring capacity, construct designs with reduced burden can be identified and shown to predictably outperform less efficient designs, despite having equivalent expression outputs.

scholarly journals Characterization of the Human Artemis Promoter by Heterologous Gene Expression In Vitro and In Vivo

2011 ◽  
Vol 30 (10) ◽  
pp. 751-761 ◽  
Author(s):  
Megan M. Multhaup ◽  
Sweta Gurram ◽  
Kelly M. Podetz-Pedersen ◽  
Andrea D. Karlen ◽  
Debra L. Swanson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Heterologous Gene Expression ◽  
Heterologous Gene

scholarly journals Cell-free prediction of protein expression costs for growing cells

2017 ◽  
Author(s):  
Olivier Borkowski ◽  
Carlos Bricio ◽  
Michaela Murgiano ◽  
Guy-Bart Stan ◽  
Tom Ellis
Keyword(s):  
Gene Expression ◽  
Host Cells ◽  
Translation Efficiency ◽  
Heterologous Proteins ◽  
E Coli ◽  
Multiple Processes ◽  
Significant Burden ◽  
The Cost ◽  
The Relationship

Translating heterologous proteins places significant burden on host cells, consuming expression resources leading to slower cell growth and productivity. Yet predicting the cost of protein production for any gene is a major challenge, as multiple processes and factors determine translation efficiency. Here, to enable prediction of the cost of gene expression in bacteria, we describe a standard cell-free lysate assay that determines the relationship betweenin vivoand cell-free measurements and γ, a relative measure of the resource consumption when a given protein is expressed. When combined with a computational model of translation, this enables prediction of thein vivoburden placed on growingE. colicells for a variety of proteins of different functions and lengths. Using this approach, we can predict the burden of expressing multigene operons of different designs and differentiate between the fraction of burden related to gene expression compared to action of a metabolic pathway.

scholarly journals Engineered Salmonella allows real-time heterologous gene expression monitoring within infected zebrafish embryos

2012 ◽  
Vol 157 (3) ◽  
pp. 413-416 ◽  
Author(s):  
Carlos Medina ◽  
Eduardo Santero ◽  
Jose Luis Gómez-Skarmeta ◽  
Jose Luis Royo
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Heterologous Gene Expression ◽  
Heterologous Gene ◽  
Zebrafish Embryos

scholarly journals Optimization of Heterologous Gene Expression for In Vitro Evolution

BioTechniques ◽  
2001 ◽  
Vol 30 (3) ◽  
pp. 474-476 ◽  
Author(s):  
Ichiro Matsumura ◽  
Mark J. Olsen ◽  
Andrew D. Ellington
Keyword(s):  
Gene Expression ◽  
Heterologous Gene Expression ◽  
Heterologous Gene ◽  
In Vitro Evolution

scholarly journals The CD11b promoter directs high-level expression of reporter genes in macrophages in transgenic mice [published erratum appears in Blood 1995 Apr 1;85(7):1983]

Blood ◽  
1995 ◽  
Vol 85 (2) ◽  
pp. 319-329 ◽  
Author(s):  
S Dziennis ◽  
RA Van Etten ◽  
HL Pahl ◽  
DL Morris ◽  
TL Rothstein ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Transgene Expression ◽  
Heterologous Gene Expression ◽  
Myeloid Cells ◽  
Reporter Genes ◽  
Regulatory Elements ◽  
Specific Expression ◽  
High Level

Abstract CD11b is the alpha chain of the Mac-1 integrin and is preferentially expressed in myeloid cells (neutrophils, monocytes, and macrophages). We have previously shown that the CD11b promoter directs cell-type- specific expression in myeloid lines using transient transfection assays. To confirm that these promoter sequences contain the proper regulatory elements for correct myeloid expression of CD11b in vivo, we have used the -1.7-kb human CD11b promoter to direct reporter gene expression in transgenic mice. Stable founder lines were generated with two different reporter genes, a Thy 1.1 surface marker and the Escherichia coli lacZ (beta-galactosidase) gene. Analysis of founders generated with each reporter demonstrated that the CD11b promoter was capable of driving high levels of transgene expression in murine macrophages for the lifetime of the animals. Similar to the endogenous gene, transgene expression was preferentially found in mature monocytes, macrophages, and neutrophils and not in myeloid precursors. These experiments indicate that the -1.7 CD11b promoter contains the regulatory elements sufficient for high-level macrophage expression. This promoter should be useful for targeting heterologous gene expression to mature myeloid cells.

Production of foreign proteins in the yeast Pichia pastoris

1995 ◽  
Vol 73 (S1) ◽  
pp. 891-897 ◽  
Author(s):  
James M. Cregg ◽  
David R. Higgins
Keyword(s):  
Gene Expression ◽  
Pichia Pastoris ◽  
Heterologous Gene Expression ◽  
Expression System ◽  
Methylotrophic Yeast ◽  
Culture Broth ◽  
Heterologous Gene ◽  
Foreign Gene ◽  
Heterologous Proteins ◽  
Yeast Pichia Pastoris

The methanol-utilizing yeast Pichia pastoris has been developed as a host system for the production of heterologous proteins of commercial interest. An industrial yeast selected for efficient growth on methanol for biomass generation, P. pastoris is readily grown on defined medium in continuous culture at high volume and density. A unique feature of the expression system is the promoter employed to drive heterologous gene expression, which is derived from the methanol-regulated alcohol oxidase I gene (AOX1) of P. pastoris, one of the most efficient and tightly regulated promoters known. The strength of the AOX1 promoter results in high expression levels in strains harboring only a single integrated copy of a foreign-gene expression cassette. Levels may often be further enhanced through the integration of multiple cassette copies into the P. pastoris genome and strategies to construct and select multicopy cassette strains have been devised. The system is particularly attractive for the secretion of foreign-gene products. Because P. pastoris endogenous protein secretion levels are low, foreign secreted proteins often appear to be virtually the only proteins in the culture broth, a major advantage in processing and purification. Key words: heterologous gene expression, methylotrophic yeast, Pichia pastoris, secretion, glycosylation.

Can heterologous gene expression shed (a torch) light on protein function?

2004 ◽  
Vol 22 (11) ◽  
pp. 557-559 ◽  
Author(s):  
Pascal Dubessay ◽  
Michel Pagès ◽  
Frédéric Delbac ◽  
Patrick Bastien ◽  
Christian Vivares ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Function ◽  
Heterologous Gene Expression ◽  
Heterologous Gene
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