V14 Comprehensive Strain Libraries of the Archaeon Thermococcus kodakarensis (1)
A multi-stage platform has been established to generate the necessary reagents to complete the
construction of ~4,600 strains. Standard molecular biology techniques are used to generate plasmid
constructs (shown above) that are verified in E. coli, then large quantities of plasmid DNA are easily
recovered for transformation into T. kodakarensis. Transformation of plasmids into T. kodakarensis are
carried out in an anaerobic chamber and resultant colonies are recovered in ~48 hours. Isolation of
genomic DNA permits diagnostic PCR confirmation of intermediate strains. Confirmed intermediate
strains are then plated under conditions that force excision of the integrated plasmid, resulting in the
modification or deletion of the target locus.
The underlying genetic technologies are based on a positive selection and counterselection. The
positive selection in based on agmatine prototrophy, whereas the counterselectable marker is based on
sensitivity to 6-methyl purine.
Comprehensive Strain Libraries of the archaeon Thermococcus kodakarensis
Archaea: Background and
Archaea are single-celled microbes
that inhabit a wide range of marine
and terrrestrial environments. Like
bacteria, Archaea lack a nucleus but
this phenotypic resemblance
undermines the closer relationship
of archaea and eukaryotes. The
challenges of culturing many
species in the laboratory have
limited progress in developing
genetic systems to understand
archaeal physiology. . For example,
requires an anaerobic environment
at a temperature of 85 ⁰C in order to
We are focused on the construction of two comprehensive strain
libraries for Thermoccocus kodakarensis. One library will contain a
collection of strains (~2,300 unique strains) wherein each non-
essential gene has been individually and markerlessly deleted. The
second collection will contain isogenic strains wherein individual
genes have been modified to encode protein products with affinity and
Thermococcus kodakarensis is a hyperthermophilic
archaeon. The Thermococcus kodakarensis genome
contains 2.09 million base pairs (bp) and is predicted
to have approximately 2306 genes. It is found
inhabiting marine hydrothermal vents and hot sulfur
springs. T. kodakarensis tends to use sulfur to grow
but in the absence of sulfur can be forced to utilize
other pathways for metabolism.
T. kodakarensis used in the lab for construction of the
libraries is the modified strain, termed TS559. T.
kodakarensis has emerged as a premier model
system for studies of archaea, due to the prominence
of the natural competence of T. kodakarensis and the
comprehensive and rapid techniques that are
available for use with its genome.
Results and Progress to Date
The final libraries will consist of ~4600 variations to strain TS559. Many reagents
are required to generate the final libraries, including ~12,000 plasmids.
“A” plasmids are used as a template to make the “B”, “C”, and “D” plasmids. The
“A” plasmids are 100% complete, thanks to Reeve/Santangelo lab at Ohio State
The “B” plasmids are ~75% complete and the “C”/”D” plasmids are ~25% complete.
~600 deletions have been confirmed and another ~300 genes have been deemed
essential (based on our statistics).
And out of another 2306 strains, around 300 tags and 330 essentials were created
from the C/D plasmids. The essentials contain the possible genes of interest, but
because of funding restrictions each and every strain cannot be checked.
Utility of the libraries
We continue the process of completing work on the ~4600 strains in order to
form the comprehensive strain libraries for T. kodakarensis.
Over 200 unique deletions strains have been shipped worldwide and used in
genetics laboratories across the world. The resulting work has established the
role of factors in DNA replication, transcription, translation, recombination, DNA
repair, RNA processing, central metabolism, etc. Over 100 tagged strains have
been shipped worldwide, and the resultant studies have determined the in vivo
associations of factors involved in RNA processing, transcription, translation,
and replication. Many studies using the fully constructed strains are in
This work is supported by funding from the National Institutes of Health to
TJS (GM100329 and GM098176). We thank all the members of the
Santangelo lab (both at CSU and OSU) for reagents, advice, and support.
We particularly thank Rie Matsumi and Lubka Cubonova for their
assistance in preparing A, B, and C/D plasmids.
Model organisms are typically used to understand shared physiology and
metabolisms. The development of many tools and techniques for these model
organisms generally speeds progress for entire communities of researchers.
Eukaryotes and Bacteria have a multitude of model systems, whereas few model
systems, if any, have been established for the Archaea. This dearth is partly due to the
challenging culturing conditions of many species and the lack of genetic tools for
manipulation of the genome. Thermococcus kodakarensis is an anaerobic,
hyperthermophilic archaea that can be easily grown and incubated in a laboratory
environment, and for which a host of genetic tools have been developed that facilitate
rapid and directed changes to the genome. Comprehensive strain libraries wherein
every gene of T. kodakarensis is individually deleted or tagged are under construction
and will provide a global resource to advance studies on archaeal and shared
archaeal-eukaryotic chemistries. An overview of the technologies employed and
results to date will be presented.
Hallie P. Febvre, Kyle R. Byrne, Adam D. Gibbons, Nicholas Gunderson, Mikaela Henry, Gladys Hill, Jesse Lindeman, Erin Lynch, Melisa Nguyen, Brett W. Burkhart, Thomas J. Santangelo
Department of Biochemistry and Molecular Biology, Colorado State University