Table 1.
Bacterial strains, plasmids, and primers used in this study
| Strain, plasmid, or primer | Relevant characteristic(s) or sequence (5′ to 3′) | Source or reference |
|---|---|---|
| Strains | ||
| E. coli DH10B | F− mcrAΔ mrr-hsdRMS mcrBC ϕ80dlacZΔM15 lacX74 deoR recA1 araD139Δ ara leu7697 galU galK ΔrpsL endA1 nupG | Gibco/BRL |
| C. perfringens | ||
| HN13 | Strain 13; ΔgalKT | 24 |
| WH1 | Strain HN13 with deletion of the region from bp 18 to 555 in cpb | This study |
| Plasmids | ||
| pGEM-T Easy | PCR cloning vector; ampicillin resistance | Promega |
| pKRAH1 | Contains bgaR-PbgaL; chloramphenicol resistance | 25 |
| pCM-GALK | Contains a Clostridium beijerinckii galK gene under the control of a ferredoxin promoter from C. perfringens | 24 |
| pET22b | Protein production vector | Novagen |
| pCPE0413 | cpb gene in pET22b | This study |
| pWH5 | pGEM-T Easy with overlapping PCR product of DNA flanking the cpb gene | This study |
| pWH6 | pCM-GALK with cpb deletion fragment | This study |
| pWH7 | pGEM-T Easy with cpb gene from HN13 | This study |
| pWH8 | pKRAH1 with cpb gene from HN13 | This study |
| Primers | ||
| CA forward | TTTTTTCATATGGAAAAGAATGTGAGAAGACTAGAAG | |
| CA reverse | TTTTTTAAGGTTATTCGTATCCATTTACTATAACCTTAAGTTCTCC | |
| OWH9 | GTCGACTTCACAAATCAATAGAAGAAATTGAAG | |
| OWH10 | GTTTTTTAATTTTTATTTTATTCGTATCCATTCACATTCTTTTCCATACTAAATACC | |
| OWH11 | GGTATTTAGTATGGAAAAGAATGTGAATGGATACGAATAAAATAAAAATTAAAAAAC | |
| OWH12 | GGATCCCTTTTTGTTTATTTTCAATTCCAAATATAC | |
| OWH13 | AATGAAAAAGGAGATAAAACTCCAG | |
| OWH14 | CCATATACAGGACTAATTGGTCC | |
| OWH15 | GTCGACGTGGGTTTAAAGTAAAGACTTTAAATC | |
| OWH24 |