Genome engineering Escherichia coli to enable hybrid prokaryotic-eukaryotic systems.
Abstract
The ability to design and engineer hybrid prokaryotic-eukaryotic systems that blend the advanced and flex flexible synthetic biology toolkits available in model prokaryotes with the genetic processing power and host relevance of various eukaryotes represents a new frontier in synthetic biology, with broad applications across biotechnology in general (Schwartz and Dayhoff 1978; Anderson et al. 2006; Sagan 1967; Leaver et al. 2009; et al. 1998). Such hybrid systems will require genetic systems for maintaining stable co-cultures, controlled invasion of prokaryotes into e eukaryotic cells, intracellular replication and infom1ation processing, as well as extrusion and recovery of prokaryotic cells. While capabilities along several of these lines have been demonstrated (Anderson et al. 2006; Grillot-Courvalin et al. 1998; lsberg et al. 1987; Miskinyte and Gordo 2013; Koli et al. 2011 ), no prior studies have focused on the use of cell-wall deficient "L-form" bacteria for this purpose, even though though is form is believed to represent an evolved strategy for supporting naturally occurring hybrid systems and it is possible to engineer bacteria to convert to L-form in a controlled manner (Leaver et al. 2009; Klieneberger 1935). Importantly, improved understanding of the genetic drivers of "L-form" formation and replication is required, as only a handful of mutations mutations have previous been identified( Glover et a l. 2009; Joseleau-Petit et al. 2007; Leaver et al. 2009). The overall objective of this proof-of-concept study is to comprehensively map mutations resulting in the stable formation and replication of L-form Escherichia coli. To do so, we will utilize a new genome-wide mapping technology developed in the Gill lab (A. D. Garst et al. 2016) to design, construct, and quantify the affect of 100,000 targeted mutations on the generation of L-fom1 E.coli. These data will not only address a key concern for the use of "L- form" bacteria but also set the stage for future expansion of the CREATE engineering platform to the hybrid system cycle envisioned herein.
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- May 07, 2018
- Source ID
- W911NF1710341
Entities
People
- Luis Serrano Pubull
Organizations
- Army Contracting Command
- Centre for Genomic Regulation
- United States Army