15Synthetic Biology: Parts, Devices and Applications, First Edition. Edited by Christina Smolke.
© 2018 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2018 by Wiley-VCH Verlag GmbH & Co. KGaA.
2
Trackable multiplex recombineering (TRMR) allows researchers to explore the
otherwise large mutational space of the Escherichia coli genome efficiently. This
method is used to simultaneously change the expression level of every gene in
the genome, so that each gene is either overexpressed or switched off. A variation
on TRMR, tunable trackable multiplex recombineering (T^2 RMR), allows expres-
sion levels to be tuned over a 10^4 -fold range. TRMR and T^2 RMR therefore allow
bacterial responses to be tuned to different environmental cues. Additionally, the
genomic changes can be tracked and identified for population dynamic studies
and for further analyses thanks to “barcoding” (or “tagging”) of every mutation.
The TRMR and T^2 RMR procedures include library design, production, and
amplification, followed by the insertion of the DNA library into a precise loca-
tion in the genome via phage-enabled homologous recombination. Then, the
heterogeneous bacterial population is subjected to a defined stress or screened
for a specific trait. Finally, beneficial mutations are identified by means of bar-
code hybridization to a microarray or by sequencing. Importantly, TRMR- and
T^2 RMR-based populations can be established by a single scientist in a single day,
and depending on the desired trait, genome-wide mapping results may be
obtained as shortly as within a week.
2.1 Introduction
While traditional engineering usually involves the design and production of
mechanical structures and devices, biological engineering is focused on modify-
ing the natural world and adapting it to human needs. Although many consider
biological engineering to be a new field, it is as old as civilization itself. Selective
breeding of plants and animals for specific traits exemplifies one of the hallmarks
of biological engineering and evolution in general: selection of a successful sub-
population that will establish the next generations, thus continuously refining
2.3 Trackable Multiplex Recombineering
and Next-Generation Genome Design Technologies:
Modifying Gene Expression in E. coli by Inserting
Synthetic DNA Cassettes and Molecular Barcodes
Emily F. Freed^1 , Gur Pines2,3, Carrie A. Eckert1,3, and Ryan T. Gill2,3
(^1) Biosciences Center, National Renewable Energy Laboratory, 15013 Denver West Parkway, Golden, CO 80401, USA
(^2) University of Colorado, Chemical and Biological Engineering, 3415 Colorado Ave, Boulder, CO, 80303 USA
(^3) University of Colorado, Renewable and Sustainable Energy Institute, 4001 Discovery Dr, Boulder, CO 80303 USA