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

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.

Keywords: Archaea; Cloning; Gene cloning and expression; Genetics of extremophiles; Hyperthermophiles; Molecular biology; Molecular biology of archaea.

Fig. 1

a Schematic diagrams of pTN1-family plasmids. Predicted ORFs are indicated by gray blocks. ORFs encoding predicted replication-associated proteins are black. In the case of T. prieurii, the complete Rep gene interrupts another Rep-like gene colored in gray with black stripes. b Phylogenetic relationship between pTN1-family Rep proteins. Unrooted phylogenetic tree generated with the full-length Rep proteins of the 4 pTN1-family plasmids

Fig. 2

Plasmid maps of pTPTK1, pTPTK2 and pTPTK3. ORFs/genetic elements are indicated by white boxes outlined in black with labels indicating the nature of each element. Gray regions inside the plasmid map indicate the source and nature of each plasmid region

Fig. 3

Digestion and gel electrophoresis of pTP2-derived plasmids. a Plasmids digested with RruI, recognizing a single site on each plasmid. Lane 1: pTPTK1 isolated from E. coli, lane 2: pTPTK3 isolated from E. coli, lane 3: pTPTK2 isolated from E. coli, lane 4: GeneRuler 1 kb DNA ladder, lane 5: pTPTK1 isolated from T. kodakarensis, lane 6: pTPTK3 isolated from T. kodakarensis, lane 7: pTPTK2 isolated from T. kodakarensis. b Plasmids digested with HindIII, recognizing multiple sites on each plasmid. Lane 1: pTPTK1 isolated from E. coli, lane 2: pTPTK3 isolated from E. coli, lane 3: pTPTK2 isolated from E. coli, lane 4: GeneRuler 1 kb DNA ladder, lane 5: pTPTK1 isolated from T. kodakarensis, lane 6: pTPTK3 isolated from T. kodakarensis, lane 7: pTPTK2 isolated from T. kodakarensis

Fig. 4

Digestion and gel electrophoresis of plasmids from double transformants. a Plasmids digested with RruI, recognizing a single site on each plasmid. Lane 1: pTNTrpE isolated from E. coli, lane 2: pTPTK1 isolated from E. coli, lane 3: GeneRuler 1 kb DNA ladder, lane 4: pTNTrpE + pTPTK1 isolated from T. kodakarensis double transformant. b Plasmids digested with RruI, recognizing a single site on each plasmid. Lane 1: pTNAg isolated from E. coli, lane 2: pTPTK2 isolated from E. coli, lane 3: GeneRuler 1 kb DNA ladder, lane 4: pTNAg + pTPTK2 isolated from T. kodakarensis double transformant