SHARK: A Specialized Host for Assembling R6K Plasmids

R6K plasmids are
commonly used for a wide range of genome engineering
applications due to their ability to support transient delivery of
genetic cargos in many hosts. The maintenance of R6K plasmids requires
specific strains. Unfortunately, many of these have obscure backgrounds,
limited availability and were not built for efficient cloning. To
address this issue, we present the construction and characterization
of a series of Pir E. coli strains
called SHARK that are built from the DH10B derivative, Marionette-Clo.
All SHARK strains have a genome encoded pir gene
for stable R6K plasmid maintenance and a λCI gene for tight unconditional repression of specific genes on plasmids.
We show that SHARK strains are >100-fold more efficient than a
commercial
Pir strain when transformed with large and complex cloning reactions.
SHARK is intended to help facilitate the cloning of R6K plasmids for
challenging genome engineering projects, with all strains and genetic
tools for their assembly being made publicly available.