title: Selection of Escherichia coli heat shock promoters toward their application as stress probes.
abstract: The mechanism of heat shock response of Escherichia coli can be explored to program novel biological functions. In this study, the strongest heat shock promoters were identified by microarray experiments conducted at different temperatures (37°C and 45°C, 5min). The promoters of the genes ibpA, dnaK and fxsA were selected and validated by RT-qPCR. These promoters were used to construct and characterize stress probes using green fluorescence protein (GFP). Cellular stress levels were evaluated in experiments conducted at different shock temperatures during several exposure times. It was concluded that the strength of the promoter is not the only relevant factor in the construction of an efficient stress probe. Furthermore, it was found to be crucial to test and optimize the ribosome binding site (RBS) in order to obtain translational efficiency that balances the transcription levels previously verified by microarrays and RT-qPCR. These heat shock promoters can be used to trigger in situ gene expression of newly constructed biosynthetic pathways.
authors.affiliation: Centre of Biological Engineering, University of Minho, 4710-057 Braga, Portugal; MIT-Portugal Program, Cambridge, MA, USA; MIT-Portugal Program, Lisbon, Portugal.
authors.forename: Joana L
authors.initials: JL
authors.lastname: Rodrigues
authors.affiliation: Centre of Biological Engineering, University of Minho, 4710-057 Braga, Portugal.
authors.forename: Marta
authors.initials: M
authors.lastname: Sousa
authors.affiliation: Department of Chemical Engineering, Synthetic Biology Engineering Research Center (SynBERC), Massachusetts Institute of Technology, Cambridge, MA 02139, USA; MIT-Portugal Program, Cambridge, MA, USA; MIT-Portugal Program, Lisbon, Portugal.
authors.forename: Kristala L J
authors.initials: KL
authors.lastname: Prather
authors.affiliation: Centre of Biological Engineering, University of Minho, 4710-057 Braga, Portugal.
authors.forename: Leon D
authors.initials: LD
authors.lastname: Kluskens
authors.affiliation: Centre of Biological Engineering, University of Minho, 4710-057 Braga, Portugal; MIT-Portugal Program, Cambridge, MA, USA; MIT-Portugal Program, Lisbon, Portugal. Electronic address: lrmr@deb.uminho.pt.
authors.forename: Ligia R
authors.initials: LR
authors.lastname: Rodrigues
mesh_terms: D004926:Escherichia coli / Q000235:genetics / Q000502:physiology*; D005798:Genes, Bacterial; D018869:Heat-Shock Response; D015335:Molecular Probes; D020411:Oligonucleotide Array Sequence Analysis; D013312:Stress, Physiological
chemical_list: D015335:Molecular Probes
keywords: E. coli; Heat shock promoters; Microarrays; RT-qPCR; Stress probes