TABLE 1.
Plasmids and oligonucleotide primers used in this study
| Plasmid or primer | Phenotype or sequencea | Source or reference |
|---|---|---|
| Plasmids | ||
| pSECE1 | Wide-host-range vector, erythromycin resistance marker | 25 |
| pCUS | Secretion vector containing signal sequence of amyA gene, derivative of pSECE1 | 18 |
| pCUSαA | Vector for secreting α-amylase, derivative of pCUS | 18 |
| pAmyA-MCS | Vector for secreting α-amylase, cloning sites BamHI-XhoI-HindIII downstream of amyA gene, derivative of pCUS | This study |
| pMCS-AmyA | Vector for secreting α-amylase, cloning sites BamHI-SpeI-XbaI-XhoI upstream of amyA gene, derivative of pCUS | This study |
| pUC19 | Cloning vector, ampicillin resistance marker | Takara Bio |
| pUC19-CA | Vector containing cA from acmA, derivative of pUC19 | This study |
| pUC19-CA2 | Vector containing two cA repeats, derivative of pUC19-CA | This study |
| pUC19-CA3 | Vector containing three cA repeats, derivative of pUC19-CA2 | This study |
| pCA-AmyA | Vector for display of α-amylase to C terminus of cA, derivative of pMCS-AmyA | This study |
| pCA2-AmyA | Vector for display of α-amylase to C terminus of two cA repeats, derivative of pMCS-AmyA | This study |
| pCA3-AmyA | Vector for display of α-amylase to C terminus of three cA repeats, derivative of pMCS-AmyA | This study |
| pAmyA-CA3 | Vector for display of α-amylase to N terminus of three cA repeats, derivative of pAmyA-MCS | This study |
| Oligonucleotide primers | ||
| amyA_F2 | 5′-CCCGATATCGATGAACAAGTGTCAATGAAAGATGGTA−3′ | |
| amyA_F3 | 5′-CCGCTCGAGGATGAACAAGTGTCAATGAAAGATGGTACG−3′ | |
| amyA_R | 5′-CCCAAGCTTATTTTAGCCCATCTTTATTATAGTTTCCAG−3′ | |
| amyA_R2 | 5′-CCGCTCGAGTTAGGATCCTTTTAGCCCATCTTTATTATAGTTTCCAG-3′ | |
| cA_F | 5′-CGCGGATCCACTAGTGTCGACTCTTCTGCTGGTACTTCTAATTCCGGTGG−3′ | |
| cA_R | 5′-ACATGCATGCCTCGAGTTTAATACGAAGATATTGACCAATTAAAATGG−3′ |
a
For primers, restriction enzyme sites are underlined.