(b) GKL reverse primer: 5^0 -CCGACCTTACAAGGATCCT-
CAAGCCGAGAACAGCGCC-3^0.
(c) T7 Pro primer: 5^0 -CGAAATTAATACGACTCACTA-
TAGG-3^0.
(d) T7 Term primer: 50 -GCTAGTTATTGCT-
CAGCGGTGGCAGC-3^0.
(e) Lux forward primer: 5^0 -GAGAGACTCGAGT-
TAATTTTTAAAGTATGGGC-3^0.
(f) Lux reverse primer: 5^0 -CTCTCTGGTACCTCAACTAT-
CAAACGCTTCGG-3^0.
(g) Gm-up primer: 5^0 -TGGAGCAGCAACGATGTTAC-3^0.
(h) Gm-downprimer:5^0 -TGTTAGGTGGCGGTACTTGG-3^0.- Thermocycler.
- DNA ladder.
- Electrophoresis apparatus for DNA agarose gel.
- Electrophoresis apparatus for SDS-PAGE analysis.
- DNA and protein ladders.
- Enzymes (with appropriate buffers): T4 DNA Ligase, NdeI,
BamHI, XhoI, KpnI, Mutazyme II (Agilent). - Plasmids: pET15b plasmid (Novagen); pUC18R6K-mini-
Tn7T-GmRplasmid [1]; pTNS2 plasmid [1]; pBAD33 [8].
29.E. coliBL21 (DE3) cells (Novagen).
30.E. coliXL1 Blue cells (Stratagene). - Refrigerated centrifuge.
- Shaking incubator.
- 200μl, 1.5 ml, and 50 ml tubes.
- 2 l conic flasks.
- Electroporation cuvettes and apparatus.
3 Methods
3.1 Amplification
and Cloning of thegkl
Lactonase Gene
This method describes amplification and cloning of thegkllacto-
nase gene from the genomic DNA isolated fromG. kaustophilus
HTA426 (GI: 56420041).- Prepare a standard PCR reaction mixture (50μl) containing
800 nM of the oligonucleotides GKL forward primer and GKL
reverse primer, and 10 ng of genomic DNA. - Amplify the gene in a thermocycler with the following para-
meters: 98C for 2 min followed by 30 cycles of 98C for 10 s,
314 Maybelle Kho Go et al.