High-throughput gene expression analysis at the level of single proteins using a microfluidic turbidostat and automated cell tracking.
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Ullman G, Wallden M, Marklund EG, Mahmutovic A, Razinkov I, Elf J
Philos. Trans. R. Soc. Lond., B, Biol. Sci. 368 (1611) 20120025 [2013-02-05; online 2012-12-24]
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.
PubMed 23267179
DOI 10.1098/rstb.2012.0025
Crossref 10.1098/rstb.2012.0025
pii: rstb.2012.0025
pmc: PMC3538428