1. Archaeal genomes encode a wide range of chemistries with promising roles in the
biotechnology marketplace. The thermostable enzymes derived from hyperthermophilic archaea
are of particular interest and the identification, characterization, and manipulation of the genes
encoding such enzymes has received a great deal of attention. As more work is performed
directly in archaeal organisms, more advanced techniques are necessary for genetic
manipulations appropriate for the organisms under study. Thermococcus kodakarensis readily
incorporates foreign DNA into its genome and has emerged as a model system for archaeal
research. T. kodakarensis has also received attention as a tractable expression platform for
hyperthermophilic enzymes that are poorly expressed in bacterial hosts or for which
incorporation of appropriate metal co-factors is essential for activity.
T. kodakarensis has no naturally occurring replicative vectors. Currently, one vector has
been designed and made available for shuttling between T. kodakarensis and Escherichia coli
(pLC70). To allow for more advanced genetic techniques, construction of additional shuttle
vectors with independent selectable markers are needed. Environmental plasmids have been
identified and sequenced from organisms that are closely related to T. kodakarensis, all of
which are hyperthermophilic and include their own specific replicative origin proteins. Two
different plasmids with the same origin of replication cannot be maintained in the same strain,
thus additional sources of natural replicative origins are needed for the development of more
shuttle vectors. Each of these environmental plasmids serves as a potential source of novel
replicative origins for use in the development of a shuttle vector for T. kodakarensis. Attempts to
develop an additional shuttle vector that replicates in T. kodakarensis have had limited
progress, due to the low copy number plasmid DNA, and low yields when attempting to isolate
the naturally occurring plasmids. To circumvent these problems, we aim to develop a new
technology that will allow for repeated isolation of the environmental plasmids as well as allow
for an in vivo assessment of replicative capabilities of each plasmid in T. kodakarensis.
In order to purify larger amounts of environmental plasmids and test their replicative
ability in T. kodakarensis, a transposon based technique will be the best methodology to
employ. If a customized transposon contains an E. coli replicative origin (p15a),
chloramphenicol resistance gene, a T. kodakarensis selectable marker (TK0149), Xho1
restriction enzyme cut sites, and the mosaic ends for efficient transposition, then environmental
plasmid DNA can be isolated (with potential of being a new shuttle vector).

2. Since purification of large amount of the naturally occurring plasmids is difficult, the
transposition reaction will take place in a mix of plasmid and genomic DNA which has been
prepped in mass quantity. The transposon DNA will randomly insert into the plasmid DNA. This
DNA will then be transformed into E. coli where it will replicate and large amounts of plasmid
DNA will be recovered. The plasmids with the transposon insertion can be directly transformed
into T. kodakarensis and the ability of plasmids to replicate in T. kodakarensis can be evaluated.
Additionally, the purified plasmids can be used as a PCR template or for molecular cloning
which will contribute to furthering our understanding of plasmid biology in archaeal organisms.

3. Works Cited
1. Berkner S, Grogan D, Albers S, Lipps G. Small multicopy, non-integrative shuttle vectors
based on the plasmid pRN1 for Sulfolobus acidocaldarius and Sulfolobus solfataricus,
model organisms of the (cren-)archaea. Nucleic Acids Research. 2007;35(12):e88-e88.
doi:10.1093/nar/gkm449.
2. Gonnet M, Erauso G, Prieur D, Le Romancer M. pAMT11, a novel plasmid isolated from
a Thermococcus sp. strain closely related to the virus-like integrated element TKV1 of
the Thermococcus kodakaraensis genome. Research in Microbiology. 2011;162(2):132-
143. doi:10.1016/j.resmic.2010.11.003.
3. Santangelo T, Cubonova L, Reeve J. Shuttle Vector Expression in Thermococcus
kodakaraensis: Contributions of cis Elements to Protein Synthesis in a
Hyperthermophilic Archaeon. Applied and Environmental Microbiology.
2008;74(10):3099-3104. doi:10.1128/aem.00305-08.
4. Soler N, Marguet E, Cortez D et al. Two novel families of plasmids from
hyperthermophilic archaea encoding new families of replication proteins. Nucleic Acids
Research. 2010;38(15):5088-5104. doi:10.1093/nar/gkq236.