Supporting information for Meuer et al. (April 2, 2002) Proc. Natl. Acad. Sci. USA, 10.1073/pnas.072615499.
Table 4.
Plasmids used in the studyPlasmid | Description and/or construction* | Source (ref.) |
pBluescript SK(+) | ApR cloning vector | Stratagene |
pBluescript KS(+) | ApR cloning vector | Stratagene |
pJK41 | ApR, PmR cloning vector, R6K replicon | 1 |
pJK61 | Source of pac-ori-aph gene cassette | 2 |
pWM368 | ApR cloning vector with Methanosarcina barkeri mcrB promoter | 3 |
pWM356 | ApR cloning vector: religation pBluescript KS after cutting w/BamHI and treatment w/T4 DNA polymerase and dNTPs | This study |
pCK18 | Spe I-cut PCR product (with primers ech-for and ech-rev ) containing ech operon cloned into SpeI site of pBluescript SK | This study |
pCK20 | Head to tail insertion of BamHI/AvrII-cut ech ' (with primers ech'-for and ech'-rev) and 'ech (with primers 'ech-for and 'ech-rev) PCR products into XbaI site of pWM356 | This study |
pCK22 | D ech1 mutation plasmid: pac-ori-aph BamHI cassette of pJK61 cloned into BamHI-cut pCK20 | This study |
pMP45 | Replacement of 845-bp NdeI/BglII fragment of pWM368 with NdeI/BglII digested PCR fragment (with primers uidA-for and uidA-rev) containing the Escherichia coli uidA gene | This study |
pJK74 | Replacement of 712-bp AscI fragment of pJK41 with AscI-cut PCR product (with primers hpt-for and hpt-rev) carrying the M. barkeri hpt gene | This study |
pJK75 | Replacement of 189-bp NdeI/SphI fragment of pMP45 with NdeI/SphI-cut PCR product (with primers echp-for and echp -rev) carrying the ech promoter | This study |
pJK76 | 3.5-kbp AflII fragment of pCK18 inserted into AflII-cut pJK75 | This study |
pJK77 | Replacement of 3,056-bp KpnI/BstEII fragment of pMP45 with 2.9-kbp KpnI/BstEII fragment of pCK18 | This study |
pJK78 | hpt::ech complementation plasmid: Insertion of 6.5-kbp ApaI fragment of pJK77 (containing ech operon) cloned into Ecl136I-deleted pJK74 (removes 173-bp fragment of hpt gene) after treatment with T4 DNA polymerase and dNTPs | This study |
*Standard methods were used for all constructions (4). All DNA fragments obtained by PCR were verified by sequencing at the W. M. Keck Center for Comparative and Functional Genomics (University of Illinois). DH5a (Life Technologies, Gaithersburg, MD) was used as the host for most plasmids; DH5a /l pir (5) was used as the host for pir-dependent R6K replicons. The pac gene encodes puromycin acetyltransferase; ori is the origin of replication from plasmid R6K; aph encodes aminoglycoside phosphotransferase; hpt encodes hypoxanthine phosphoribosysltransferase; uidA encodes b-glucuronidase; ech and mcr are the ferredoxin-dependent hydrogenase and methylreductase operons, respectively, of M. barkeri.
1. Metcalf, W. W. (1999) in Methods in Microbiology: Genetic Methods for Diverse Prokaryotes, eds. Smith, M. & Sockett, L. (Academic Press, London), Vol. 29, Chap. 10.
2. Zhang, J. K., White, A. K., Kuettner, H. C., Boccazzi, P. & Metcalf, W. W. J. (2002) J. Bacteriol. 184, 1449-1454.
3. Zhang, J. K., Pritchett, M. A., Lampe, D. J., Robertson, H. M. & Metcalf, W. W. (2000) Proc. Natl. Acad. Sci. USA 97, 9665–9670.
4. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A. & Struhl, K. (1992) Current Protocols in Molecular Biology (Wiley, New York).
5. Miller, V. L. & Mekalanos, J. J. (1988) J. Bacteriol. 170, 2575–2583.