Synthetic pluripotent bacterial stem cells

Abstract

We describe a synthetic genetic circuit for controlling asymmetric cell division in E. coli in which a progenitor cell creates a differentiated daughter cell while retaining its original phenotype.Specifically, we engineered an inducible system that can bind and segregate plasmid DNA to a single position in the cell. Upon cell division, co-localized plasmids are kept by one and only one of the daughter cells. The other daughter cell receives no plasmid DNA and is hence irreversibly differentiated from its sibling. In this way, we achieved asymmetric cell division through asymmetric plasmid partitioning. We then used this system to achieve physical separation of genetically distinct cells by tying motility to differentiation. Finally, we characterized an orthogonal inducible circuit that enables the simultaneous asymmetric partitioning of two plasmid species, resulting in cells that have four distinct differentiated states. These results point the way towards engineering multicellular systems from prokaryotic hosts. biochemical or physical tasks throughout the organism. In multicellular eukaryotes, cellular differentiation is generally achieved through complex regulatory networks that utilize transcriptional regulation, post-transcriptional modifications, and chromatin remodeling 19-21 . To differentiate asymmetrically, a progenitor cell (such as a stem cell) senses chemical cues in the environment to alter the transcriptional landscape in the daughter cell. This transcriptional rearrangement of the daughter cell is complete enough that de-differentiation is rare and reproducible in the lab only through the very specific and simultaneous induction of many genes 22 .Several attempts have been made to create synthetic bacteria that have multiple stable transcriptional profiles 3,23 . The first of these was the corepressive toggle switch in E. coli designed by Gardner and colleagues 3 . However, the corepressive toggle does not create irreversibly differentiated cell types. It is sensitive to intrinsic and extrinsic sources of gene regulatory noise 3,24-27 , and can only transiently maintain one state before stochastically switching back to the other.Others have used recombinases 28 to alter DNA sequences permanently, or controllably alter the copy numbers of plasmids to affect gene regulation and cell differentiation 29,30 .Here, we repurposed two key elements of the chromosome partitioning system (par) of Caulobacter crescentus 31 to create asymmetric cell division and irreversible differentiation in E. coli. The par system is common in prokaryotes and is principally responsible for partitioning low copy number plasmids or chromosomes upon cell division 32 . The par systems generally rely on the interaction of three elements: a centromere-like cis-acting sequence, parS, present on the plasmid/chromosome; a centromere-binding trans-acting protein, ParB; and an NTPase, usually called ParA 33 . The initial step is the formation of the partition complex, in which all the copies of the plasmid or chromosomal DNA are gat...