MANIPULATING BACTERIAL GROWTH FOR SINGLE CELL GENOMICS

Los Alamos Nat Secty-Los Alamos Nat Lab, Los Alamos NM

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Abstract

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. FACS and multiple displacement amplification (MDA) are essential means in the study of single cell genomics. Where FACS detects and sorts cellular candidates from the environment, MDA randomly amplifies segments of the isolated cell's genome from minute quantities of DNA template. The products of this amplification process are then sequenced and assembled to determine the complete genomic identity of the isolated cell. As FACS and MDA are critical tools for single cell genomics, certain issues are evident that contribute to a low- efficient, high-cost, high-labor process of genomic assembly. In FACS, the differentiation of living cells from particles from a given environmental sample is vital to determine possible cellular candidates for downstream genomic studies. MDA has amplification biases based on the genomic template's GC content. Furthermore, gaps in the genomic sequence necessitates additional template to complete the sequence gaps. If the genomic template is scarce and/or the isolated cell cannot be recovered from its environment, then the cell's genome may not be completed. To address these issues, normal growth cycles of bacterial representatives Escherichia coli K12 and Bacillus subtilis were manipulated to result in phenotypes of larger-than-normal cell size and polyploidy. A larger cell suggests metabolic activity and, thus, a living cell. If such a larger cell possesses multiple copies of its genome, then the MDA and subsequent genomic assembly processes may be streamlined.