TABLE 2.
Primers used for construction and confirmation of S. agalactiae GD201008-001
| Primer | Sequence (5′→3′)a | Restriction site | Function |
|---|---|---|---|
| hylB-LF | CCGGAATTCTGATAAGAAAAGTCAATGGAC | EcoRI | Left arm of hylB; amplifies flank sequence located in coding region of hylB upstream (632 bp) |
| hylB-LRA | AGGTTTTTTATGTACGGCTAGTCTGTTGAAGCTGTAACTGAT | ||
| hylB-RFA | ATCAGTTACAGCTTCAACAGACTAGCCGTACATAAAAAACCT | Right arm of hylB; amplifies flank sequence located in coding region of hylB upstream (676 bp) | |
| hylB-RR | CGCGGATCCGAAGGACCAGGTGAAAAATTC | BamHI | |
| hylB-F | CCGTCTCTTGGGAAAAT | Fragment for hylB ORF; used to confirm deletion of hylBb | |
| hylB-R | CATGGTTTGAGGTTGGT | ||
| ChylB-F | CCGGAATTCAGGCAATTTACTATCAAAGTAGCAC | EcoRI | Fragment for complementation of hylB; amplifies structural gene portion of hylB, including its own promoter (3,382 bp) |
| ChylB-R | ATACTGCAGGATCTTCGTTACGCTATCGATTCTA | PstI |
a
Restriction sites are underlined.
b
The wild-type or complemented strain will produce a 2,334-bp fragment by PCR, but the mutant strain will not.