A series of new E. coli-Thermococcus shuttle vectors compatible with previously existing vectors

Extremophiles : Life Under Extreme Conditions
Ryan CatchpolePatrick Forterre

Abstract

Hyperthermophilic microorganisms are an important asset in the toolkits of biotechnologists, biochemists and evolutionary biologists. The anaerobic archaeon, Thermococcus kodakarensis, has become one of the most useful hyperthermophilic model species, not least due to its natural competence and genetic tractability. Despite this, the range of genetic tools available for T. kodakarensis remains limited. Using sequencing and phylogenetic analyses, we determined that the rolling-circle replication origin of the cryptic mini-plasmid pTP2 from T. prieurii is suitable for plasmid replication in T. kodakarensis. Based on this replication origin, we present a novel series of replicative E. coli-T. kodakarensis shuttle vectors. These shuttle vectors have been constructed with three different selectable markers, allowing selection in a range of T. kodakarensis backgrounds. Moreover, these pTP2-derived plasmids are compatible with the single-existing E. coli-T. kodakarensis shuttle vector, pLC70. We show that both pTP2-derived and pLC70-derived plasmids replicate faithfully while cohabitating in T. kodakarensis cells. These plasmids open the door for new areas of research in plasmid segregation, DNA replication and gene expression.

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Citations

May 12, 2018·Biotechnology and Bioengineering·Emily FreedCarrie A Eckert
Jan 24, 2019·Extremophiles : Life Under Extreme Conditions·Geraldy L S LimanThomas J Santangelo
Sep 11, 2019·Environmental Microbiology·Catherine BadelViolette Da Cunha

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Datasets Mentioned

BETA
ABR10429.1

Methods Mentioned

BETA
PCR
electrophoresis

Software Mentioned

BMGE
iTOL
IQ
TREE
ModelFinder
HHpred
MSAprobs
Phyre2
BLAST

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