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Bruneaux, Luke Julien

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Bruneaux

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Luke Julien

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Bruneaux, Luke Julien
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    Multiple Unnecessary Protein Sources and Cost to Growth Rate in E.coli
    (2013-07-25) Bruneaux, Luke Julien; Franklin, Melissa; Prentiss, Mara; Franklin, Melissa; Feldman, Gary
    The fitness and macromolecular composition of the gram-negative bacterium E.coli are governed by a seemingly insurmountable level of complexity. However, simple phenomenological measures may be found that describe its systems-level response to a variety of inputs. This thesis explores phenomenological approaches providing accurate quantitative descriptions of complex systems in E.coli. Chapter 1 examines the relationship between unnecessary protein production and growth rate in E.coli. It was previously unknown whether the negative effects on growth rate due to multiple unnecessary protein fractions would add linearly or collectively to produce a nonlinear response. Within the regime of this thesis, it appears that the interplay between growth rate and protein is consistent with a non-interacting model. We do not need to account for complex interaction between system components. Appendix A describes a novel technique for real-time measurement of messenger RNA in single living E.coli cells. Using this technique, one may accurately describe the transcriptional response of gene networks in single cells.
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    Minimally Invasive Determination of mRNA Concentration in Single Living Bacteria
    (Oxford University Press, 2008) Guet, Calin C.; Bruneaux, Luke Julien; Min, Taejin L.; Siegal-Gaskins, Dan; Figueroa, Israel; Emonet, Thierry; Cluzel, Philippe
    Fluorescence correlation spectroscopy (FCS) has permitted the characterization of high concentrations of noncoding RNAs in a single living bacterium. Here, we extend the use of FCS to low concentrations of coding RNAs in single living cells. We genetically fuse a red fluorescent protein (RFP) gene and two binding sites for an RNA-binding protein, whose translated product is the RFP protein alone. Using this construct, we determine in single cells both the absolute [mRNA] concentration and the associated [RFP] expressed from an inducible plasmid. We find that the FCS method allows us to reliably monitor in real-time [mRNA] down to »40nM (i.e. approximately two transcripts per volume of detection). To validate these measurements, we show that [mRNA] is proportional to the associated expression of the RFP protein. This FCS-based technique establishes a framework for minimally invasive measurements of mRNA concentration in individual living bacteria.