Imperial College London

ProfessorPaulFreemont

Faculty of MedicineDepartment of Medicine

Chair in Protein Crystallography
 
 
 
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Contact

 

+44 (0)20 7594 5327p.freemont

 
 
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Location

 

259Sir Alexander Fleming BuildingSouth Kensington Campus

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Summary

 

Publications

Citation

BibTex format

@article{Freemont:2018:10.1073/pnas.1715806115,
author = {Freemont, PS and Moore, S and MacDonald, J and Wienecke, S and Ishwarbhai, A and Tsipa, A and Aw, R and Kylilis, N and Bell, D and McCymont, D and Jensen, K and Polizzi, K and Biedendieck, R},
doi = {10.1073/pnas.1715806115},
journal = {Proceedings of the National Academy of Sciences},
pages = {E4340--E4349},
title = {Rapid acquisition and model-based analysis of cell-free transcription-translation reactions from non-model bacteria},
url = {http://dx.doi.org/10.1073/pnas.1715806115},
volume = {115},
year = {2018}
}

RIS format (EndNote, RefMan)

TY  - JOUR
AB - Native cell-free transcription–translation systems offer a rapid route to characterize the regulatory elements (promoters, transcription factors) for gene expression from nonmodel microbial hosts, which can be difficult to assess through traditional in vivo approaches. One such host, Bacillus megaterium, is a giant Gram-positive bacterium with potential biotechnology applications, although many of its regulatory elements remain uncharacterized. Here, we have developed a rapid automated platform for measuring and modeling in vitro cell-free reactions and have applied this to B. megaterium to quantify a range of ribosome binding site variants and previously uncharacterized endogenous constitutive and inducible promoters. To provide quantitative models for cell-free systems, we have also applied a Bayesian approach to infer ordinary differential equation model parameters by simultaneously using time-course data from multiple experimental conditions. Using this modeling framework, we were able to infer previously unknown transcription factor binding affinities and quantify the sharing of cell-free transcription–translation resources (energy, ribosomes, RNA polymerases, nucleotides, and amino acids) using a promoter competition experiment. This allows insights into resource limiting-factors in batch cell-free synthesis mode. Our combined automated and modeling platform allows for the rapid acquisition and model-based analysis of cell-free transcription–translation data from uncharacterized microbial cell hosts, as well as resource competition within cell-free systems, which potentially can be applied to a range of cell-free synthetic biology and biotechnology applications.
AU - Freemont,PS
AU - Moore,S
AU - MacDonald,J
AU - Wienecke,S
AU - Ishwarbhai,A
AU - Tsipa,A
AU - Aw,R
AU - Kylilis,N
AU - Bell,D
AU - McCymont,D
AU - Jensen,K
AU - Polizzi,K
AU - Biedendieck,R
DO - 10.1073/pnas.1715806115
EP - 4349
PY - 2018///
SN - 0027-8424
SP - 4340
TI - Rapid acquisition and model-based analysis of cell-free transcription-translation reactions from non-model bacteria
T2 - Proceedings of the National Academy of Sciences
UR - http://dx.doi.org/10.1073/pnas.1715806115
UR - http://hdl.handle.net/10044/1/59106
VL - 115
ER -