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 Information

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, Thermococcus kodakarensis requires an anaerobic environment at a temperature of 85 C in order to ⁰thrive.

Overarching Goal

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 epitope tags.

Thermococcus kodakarenesis

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

Plasmid Construction

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 University.

The “B” plasmids are ~75% complete and the “C”/”D” plasmids are ~25% complete.

Strain Construction

~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 progress.

Acknowledgments

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.

AbstractModel 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