title: High-throughput gene expression analysis at the level of single proteins using a microfluidic turbidostat and automated cell tracking.
abstract: We have developed a method combining microfluidics, time-lapsed single-molecule microscopy and automated image analysis allowing for the observation of an excess of 3000 complete cell cycles of exponentially growing Escherichia coli cells per experiment. The method makes it possible to analyse the rate of gene expression at the level of single proteins over the bacterial cell cycle. We also demonstrate that it is possible to count the number of non-specifically DNA binding LacI-Venus molecules using short excitation light pulses. The transcription factors are localized on the nucleoids in the cell and appear to be uniformly distributed on chromosomal DNA. An increase in the expression of LacI is observed at the beginning of the cell cycle, possibly because some gene copies are de-repressed as a result of partitioning inequalities at cell division. Finally, a size-growth rate uncertainty relation is observed where cells living in rich media vary more in the length at birth than in generation time, and the opposite is true for cells living in poorer media.
authors.affiliation: Department of Cell and Molecular Biology, Science for Life Laboratory, Uppsala University, 75105 Uppsala, Sweden.
authors.forename: G
authors.initials: G
authors.lastname: Ullman
authors.forename: M
authors.initials: M
authors.lastname: Wallden
authors.forename: E G
authors.initials: EG
authors.lastname: Marklund
authors.forename: A
authors.initials: A
authors.lastname: Mahmutovic
authors.forename: Ivan
authors.initials: I
authors.lastname: Razinkov
authors.forename: J
authors.initials: J
authors.lastname: Elf
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry; D002453:Cell Cycle; D058948:Cell Tracking / Q000379:methods; D002875:Chromosomes / Q000737:chemistry; D004269:DNA, Bacterial / Q000737:chemistry; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D057166:High-Throughput Screening Assays; D007091:Image Processing, Computer-Assisted; D056968:Lac Repressors / Q000737:chemistry; D016013:Likelihood Functions; D008565:Membrane Proteins / Q000737:chemistry; D000072236:Methyl-Accepting Chemotaxis Proteins; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation / Q000379:methods*; D044085:Microfluidics / Q000295:instrumentation / Q000379:methods; D009004:Monosaccharide Transport Proteins / Q000737:chemistry; D009391:Nephelometry and Turbidimetry / Q000379:methods*; D027981:Symporters / Q000737:chemistry; D059008:Time-Lapse Imaging / Q000379:methods; D014157:Transcription Factors / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D056968:Lac Repressors; C539064:LacI protein, E coli; C050221:LacY protein, E coli; D008565:Membrane Proteins; D000072236:Methyl-Accepting Chemotaxis Proteins; D009004:Monosaccharide Transport Proteins; D027981:Symporters; D014157:Transcription Factors