Highly Multiplexed Imaging of E. Coli Chromosome and Sensitive Detection of Single-Cell Protein

Abstract

The driving force for biology research is the development of new techniques which allow high-sensitivity, high-throughput measurement in various contexts. Over the past decade, the emerging of a variety of single-cell techniques have greatly transformed our understanding of biological system. My thesis work was therefore focused on development of new single- cell techniques and use the techniques to generate new insights into biological system. Specifically, in the first part of my thesis work, we developed DNA seqFISH, a technique that allows us to image more than 100 different loci on the chromosome in single cells. We applied this technique to image E. coli chromosome with 50kb genomic resolution and 50nm spatial precision. Our data allows us to parse the E. coli chromosome structure according to their different spatial conformations and different cell-cycle stages. We identified two chromosome conformations with distinct domain structures, which is obscured from previous population-average research. We further characterized the domain structure dynamics during daughter chromosome segregation. Therefore, our data provides a high- resolution, dynamic view of E. coli chromosome structure. In the second part, we developed a novel method for sensitive detection of targeted protein and its post-translational modification (PTM) isoform in single cells. Instead of depending on antibodies to distinguish targeted protein and its PTM isoform, we developed an efficient covalent barcoding strategy to barcode targeted protein inside the cells. Thereafter, targeted protein and its PTM isoform are separated by conventional gel electrophoresis, while their single-cell identity is preserved in the covalently attached oligo. By counting the attached DNA oligos using next-generation sequencing, targeted protein, and its PTM isoform can be accurately measured. We demonstrated the utility of the technology by quantification of histone protein, H2B and its mono-ubiquitination isoform, H2Bub at single-cell level. Our method revealed the single-cell heterogeneities of H2Bub/H2B ratio and its cell-cycle dynamics. Our method therefore provides an antibody-free method for sensitive detection of proteins and its isoforms in single cells.