GeneGuard: A Modular Plasmid System Designed for Biosafety

Synthetic biology
applications in biosensing, bioremediation, and
biomining envision the use of engineered microbes beyond a contained
laboratory. Deployment of such microbes in the environment raises
concerns of unchecked cellular proliferation or unwanted spread of
synthetic genes. While antibiotic-resistant plasmids are the most
utilized vectors for introducing synthetic genes into bacteria, they
are also inherently insecure, acting naturally to propagate DNA from
one cell to another. To introduce security into bacterial synthetic
biology, we here took on the task of completely reformatting plasmids
to be dependent on their intended host strain and inherently disadvantageous
for others. Using conditional origins of replication, rich-media compatible
auxotrophies, and toxin–antitoxin pairs we constructed a mutually
dependent host-plasmid platform, called GeneGuard. In this, replication
initiators for the R6K or ColE2-P9 origins are provided in
trans by a specified host, whose essential thyA or dapA gene is translocated from a genomic to
a plasmid location. This reciprocal arrangement is stable for at least
100 generations without antibiotic selection and is compatible for
use in LB medium and soil. Toxin genes ζ or Kid are also employed
in an auxiliary manner to make the vector disadvantageous for strains
not expressing their antitoxins. These devices, in isolation and in
concert, severely reduce unintentional plasmid propagation in E. coli and B. subtilis and do not disrupt
the intended E. coli host’s growth dynamics.
Our GeneGuard system comprises several versions of modular cargo-ready
vectors, along with their requisite genomic integration cassettes,
and is demonstrated here as an efficient vector for heavy-metal biosensors.