TABLE 1.
Oligonucleotide primers used in this study
| Primer | Orientation | Sequence (5′→3′)a |
|---|---|---|
| Cloning | ||
| CdxA (ORF0348) | Forward | 5′-catatgCAGGCTCCTCAGTTACGAGC-3′ |
| Reverse | 5′-ctcgagTTATTTATTTCTTCTCAATAAGTTAAGCG-3′ | |
| SDM1 | 5′-TAGGTGATAATGCTGCTCACATGGTTGGTACAGCTAAAC-3′ | |
| SDM2 | 5′-GTTTAGCTGTACCAACCATGTGAGCAGCATTATCACCTA-3′ | |
| Xyl3A (ORF0398) | Forward | 5′-GACGACGACAAGATGCTCATCTGCGCTGCTGAAAAG-3′ |
| Reverse | 5′-GAGGAGAAGCCCGGTTAATTTAACGTATAATGTATCTG-3′ | |
| Xyl3B (ORF0911) | Forward | 5′-GCGCcatatgCAAACTATACTTATTAATCAGCAGG-3′ |
| Reverse | 5′-CGCGctcgagTCACTTGATGACTTCAG-3′ | |
| Xyl3C (ORF0975) | Forward | 5′-GCGCcatatgATGAAAAGTAAACAACTAATAAC-3′ |
| Reverse | 5′-CGCGctcgagTTATTTTAGGTAAATAATTAATTTTTTC-3′ | |
| Mutagenesisb | ||
| Xyl3B E115A | Forward | 5′-GCTCTATTCCACGAAGCAGTGCTCTCGGGTGTT-3′ |
| Reverse | 5′-AACACCCGAGAGCACTGCTTCGTGGAATAGAGC-3′ | |
| Xyl3B E115D | Forward | 5′-GCTCTATTCCACGAAGATGTGCTCTCGGGTGTTAA −3′ |
| Reverse | 5′-TTAACACCCGAGAGCACATCTTCGTGGAATAGAGC −3′ | |
| Xyl3B R177A | Forward | 5′-CGAAATCCAAGTTTCAACGCGCTCGAAGAGTCGTATGG-3′ |
| Reverse | 5′-CCATACGACTCTTCGAGCGCGTTGAAACTTGGATTTCG-3′ | |
| Xyl3B K214A | Forward | 5′-GTGTGGGGGCTTGCAGCGCGCACTATCTCGGATATG-3′ |
| Reverse | 5′-CATATCCGAGATAGTGCGCGCTGCAAGCCCCCACAC-3′ | |
| Xyl3B H215A | Forward | 5′-GGGGGCTTGCAGCAAGGCCTATCTCGGATATGGT-3′ |
| Reverse | 5′-ACCATATCCGAGATAGGCCTTGCTGCAAGCCCCC-3′ | |
| Xyl3B M251A | Forward | 5′-CTGGAAGCAAAGCGCTGGCGCCTGGTTATCACGCTG-3′ |
| Reverse | 5′-CAGCGTGATAACCAGGCGCCAGCGCTTTGCTTCCAG-3′ | |
| Xyl3B D286A | Forward | 5′-TGGTATGGTGGTTAGTGCCTATACAGCCATAGACC-3′ |
| Reverse | 5′-GGTCTATGGCTGTATAGGCACTAACCACCATACCA-3′ | |
| Q-PCRc | ||
| GyrAQ-PCR | Forward | 5′-CCCGTGTAGTAGGTGAGGTTCTTG-3′ |
| Reverse | 5′-TCCAGTCTTGGCCCATACG-3′ | |
| CdxAQ-PCR | Forward | 5′-TTAGGAATTTGTTTGTCTGGAGCAT-3′ |
| Reverse | 5′-GCTTTCTCTTCCAACGTCATAGC-3′ | |
| Xyl3AQ-PCR | Forward | 5′-GGCCTGTGCCAAGCACTTT-3′ |
| Reverse | 5′-CGCGTGGCGAGATGTTATT-3′ | |
| Xyl3BQ-PCR | Forward | 5′-CGCTTCGAGGCAAACAGAAT-3′ |
| Reverse | 5′-GGGAACGAATAGTCACCACACA-3′ | |
| Xyl3CQ-PCR | Forward | 5′-AAACTCGGTGACTTTGATTCTGATAAC-3′ |
| Reverse | 5′-TTTGGTAGGCAAGCTGTTTATGTT-3′ |
a
Oligonucleotide primers were synthesized by Integrated DNA Technologies (Coralville, IA). Restriction enzyme sites are in lowercase type in the primer sequence. Underlined sequences indicate engineered codon mutations.
b
Residues were selected for conservative mutagenesis due to their conservation as predicted by an alignment of the primary amino acid sequences for family 3 glycoside hydrolases with biochemically defined catalytic activities, and primers were designed in accordance with the QuikChange protocol by Stratagene (La Jolla, CA).
c
Primers for Q-PCR were designed by using the Primer Express v3.0 program from Applied Biosystems.