TABLE 1.
Strains, plasmids, and PCR primers used in this study
| Strain, plasmid, or primer | Descriptiona | Source, reference, or ATCC no. |
|---|---|---|
| Strains | ||
| Escherichia coli | ||
| DH10B | F−mcrA Δ(mrr-hsdRMS-mcrBC) φ80dlacZΔM15 ΔlacX74 deoR recA1 | Invitrogen |
| BL21(DE3) | F−dcm ompT hsdS (rB− mB−) gal | Novagen |
| Rosetta gami(DE3) | Δara-leu7697 ΔlacX74 ΔphoAPvuII phoR araD139 ahpC galE galK rpsL F′[lac + (lacIq)pro] gor522::Tn10 (Tcr) trxB::Kan (DE3) pRARE6 (Cmr) | Novagen |
| Fibrobacter | ||
| F. succinogenes S85 | ATCC 19169 | |
| F. succinogenes A3C | ATCC 51219 | |
| F. intestinalis DR7 | ATCC 43855 | |
| F. intestinalis NR9 | ATCC 43854 | |
| Plasmids | ||
| pQE80L | Cloning and expression vector, Apr | QIAGEN |
| pET30a | Cloning and expression vector, Kmr | Novagen |
| pQE-ClCBase | Apr, cel10A gene from F. succinogenes S85 | This study |
| pQE-Cel8B | Apr, cel8B gene from F. succinogenes S85 | This study |
| pET30a-Cel9B | Kmr, cel9B gene from F. succinogenes S85 | This study |
| pET30a-Cel9BΔBTD | Kmr, cel9B gene from F. succinogenes S85, basic C-terminal domain removed | This study |
| pET30a-Cel5H | Kmr, cel5H gene from F. succinogenes S85 | This study |
| pLE69B | Apr, cel51A gene from F. succinogenes S85 | 26 |
| Primers | ||
| ClCBase-1 | TTAATGCATGCCAGTTGAACAATGGTGATATG (SphI) | This study |
| ClCBase-2C | TATTTGTCGACTTACTTCACGGTAATCTGG (SalI) | This study |
| Cel9B-P4 | ATATGGATCCGCGACTGCCTATATCAAC (BamHI) | This study |
| Cel9B-P3C | TATCTTCTCGAGTTACTTCAGATGGTTG (XhoI) | This study |
| Cel9BΔBTD-P5C | TATCTTCTCGAGTTACTTCGCAACACCCGGTG (XhoI) | This study |
| Cel8B-1 | TTAATGAGCTCGGGCGATTCCCGTTCCCGC (SacI) | This study |
| Cel8B-2C | TATTTCCCGGGTTACTTCACGTTGACGCGG (XmaI) | This study |
| Cel5H-1 | CTCTGTGGATCCGTGACTGCTAGCCGTGTTGG (BamHI) | This study |
| Cel5H-2C | ACGCTGCTCGAGTTACTTGATGGAGATACG (XhoI) | This study |
a
In sequences, the restriction sites for the endonucleases in parentheses are in boldface.