Enrique Balleza, Ph.D.

Research

My research project is to obtain, in single Escherichia coli cells, genome wide transcriptional expression patterns during exponential growth phase. This implies that I will record the transcriptional activity of all promoters as a function of time. This single cell approach allows for both the identification of local and global dynamic transcriptional features that are masked in microarrays or flow cytometry. For this assay, I am using a technology developed in the Cluzel Lab consisting in an agarose pad with printed microfeatures on it that force cells to grow in linear microcolonies. The agarose pad is contained inside a microfluidics chamber in order to expose the linear microcolonies to a constant environment. The agarose pad and the microfluidics chamber compose a microchemostat that constrain the growth of linear microcolonies. We will follow the transcriptional state of E. coli cells using as reporters promoter-GFP chromosomal insertions with kanamycin resistance markers. I am generating a library of this constructs with all the promoters with experimental evidence in E. coli using a protocol based on the lambda Red homologous recombination technique(1). Currently, I track automatically single cells in linear microcolonies during 20 hours in chemostatic conditions. To accomplish this, I have developed a probabilistic cell growth model and the corresponding Bayesian statistics. Also, for a strain with an araBAD promoter-GFP chromosomal insertion, I have demonstrated that I can perform a robust quantification of fluorescence in individual cells automatically. With the above developments, I can measure the instantaneous growth rate of individual cells in linear colonies, their fluorescence from a chromosomal integrated single copy expression reporter and, most importantly, the corresponding transcriptional activity (time derivative of fluorescence divided by the instantaneous cell length). All measurements done automatically in time periods of approximately 20 hours and in four different fields of view with a time resolution of 5 minutes. By extending the last measurements to every expression reporter I will acquire transcriptome patterns at the single cell level.