pTRA - A reporter system for monitoring the intracellular dynamics of gene expression

The presence of standardised tools and methods to measure and represent accurately biological parts and functions is a prerequisite for successful metabolic engineering and crucial to understand and predict the behaviour of synthetic genetic circuits. Many synthetic gene networks are based on transc...

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Bibliographic Details
Published in:PloS one 2018-05, Vol.13 (5), p.e0197420-e0197420
Main Authors: Wagner, Sabine G, Ziegler, Martin, Löwe, Hannes, Kremling, Andreas, Pflüger-Grau, Katharina
Format: Article
Language:English
Subjects:
Bioengineering
Biology and life sciences
Biotechnology
Blotting, Western
Circuits
Cloning
E coli
Engineering and Technology
Escherichia coli
Fluorescence
Gene expression
Genes, Reporter - genetics
Genetic engineering
Genetic Techniques
Intracellular
Measurement methods
Mechanical engineering
Metabolic engineering
Metabolism
Metabolites
Physiological aspects
Plasmids
Plasmids - genetics
Promoter Regions, Genetic - genetics
Protein Biosynthesis - genetics
Proteins
Research and Analysis Methods
RNA, Messenger - genetics
Synthetic biology
Synthetic Biology - methods
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Summary:The presence of standardised tools and methods to measure and represent accurately biological parts and functions is a prerequisite for successful metabolic engineering and crucial to understand and predict the behaviour of synthetic genetic circuits. Many synthetic gene networks are based on transcriptional circuits, thus information on transcriptional and translational activity is important for understanding and fine-tuning the synthetic function. To this end, we have developed a toolkit to analyse systematically the transcriptional and translational activity of a specific synthetic part in vivo. It is based on the plasmid pTRA and allows the assignment of specific transcriptional and translational outputs to the gene(s) of interest (GOI) and to compare different genetic setups. By this, the optimal combination of transcriptional strength and translational activity can be identified. The design is tested in a case study using the gene encoding the fluorescent mCherry protein as GOI. We show the intracellular dynamics of mRNA and protein formation and discuss the potential and shortcomings of the pTRA plasmid.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0197420