Plasmids & Vectors
Plasmid vectors and detailed instructions for subtilase expression are available on request from the Wells laboratory. Subtiligase is expressed in Bacillus subtilis and the enzyme is secreted to the media. The enzyme is purified through ammonium sulfate precipitation, anion exchange, isopropyl resin capture (for the catalytic cysteine residue in subtilase), and gel filtration. The enzyme is stored at −80 °C and retains activity for at least 2 years after purification. Activity can be tested and quantified using FRET ester reporters (Shimbo et al., 2012; Yoshihara, Mahrus, & Wells, 2008).
Shuttle Vector Systems
Plasmid vectors using puromycin resistance as a selective marker have been developed for many methanogenic Archaea. The current vectors are constructed either with autonomous replication from rolling-circle origins, or else with the ability to integrate into the chromosome using cassette markers flanked by chromosomal DNA fragments to promote homologous replication. Recently, vectors have been developed based on the term adapted hygromycin B phosphotransferase gene, which confers resistance to hygromycin at 85 °C.
Mutations in the gene that result in increased thermostability of the protein, have allowed two shuttle vectors to be developed for hyperthermophiles: pEXSs, which utilizes a phage SSV1 replication origin, can be propagated in S. solfataricus under selection for hygromycin resistance. Another autonomously replicating plasmid, pAG21, contains the ADH gene from S. solfataricus and a replication origin from the Pyrococcus abyssi plasmid pGT5. The plasmid propagates in both P. furiosus and S. solfataricus.
The selection marker used is imperfect, however. The system uses toxic alcohols such as butanol that are detoxified by the Sulfolobus adh gene encoded by the plasmid and the selection provided is weak, possibly because of resident alcohol dehydrogenases encoded by the Pyrococcus spp.
Prepare plasmid vector by fusing CAT-containing plasmid with DNA element to be tested. Grow eukaryotic cells to be transfected.3 Prepare TLC tank by placing a sheet of Whatman 3MM filter paper around the inside of the tank and add 200 ml of chloroform: methanol (19:1) solvent to equilibrate tank atmosphere. Make solvent system fresh for each use, because chloroform is very volatile.