Table 1 Bacterial strains, plasmids and primers used in this study
Strain, plasmid or primers | Description or sequence (5’-3’) | Source or purpose |
|---|---|---|
Strains | ||
Escherichia coli JM109 | F', traD36, proAB Δ. lacIq, Δ (lacZ), M15/Δ (lac-proAB), glnV44, e14−, gyrA96, recA1, relA1, endA1, thi, hsdR17 | Our lab |
Bacillus licheniformis CICIM B1391 | Wild-type | Our lab |
BLA | B. licheniformis CICIM B1391 harboring plasmid pKA | This work |
BLAR | B. licheniformis CICIM B1391 harboring plasmid pKAR | This work |
BLAR2 | B. licheniformis CICIM B1391 harboring plasmid pKAR2 | This work |
BLAR3 | B. licheniformis CICIM B1391 harboring plasmid pKAR3 | This work |
BLAR4 | B. licheniformis CICIM B1391 harboring plasmid pKAR4 | This work |
BLAR5 | B. licheniformis CICIM B1391 harboring plasmid pKAR5 | This work |
BLKA | B. licheniformis CICIM B1391, ΔamyL | This work |
BLPE | B. licheniformis CICIM B1391 harboring plasmid pHY300-Prha-eGFP | This work |
Plasmids | ||
pMD19-T | E. coli cloning vector, ApR | TaKaRa |
pHY300-PLK | E. coli/Bacillus shuttle vector, Ampr /Tetr | Our lab |
pHY300-P2-eGFP | pHY, with the eGFP cassette mediated by P2 promoter | Our lab |
pHY300-Prha-eGFP | pHY, with the eGFP cassette mediated by Prha promoter | This work |
pNZTT-AFKF | pNZTT, with the deletion cassette of amyL | Our lab |
pKA | pHY-PLK300, with the deletion cassette of amyL | This work |
pKAR | pKA, with the expression cassette of recombinase BPR1 | This work |
pKAR2 | pKA, with the expression cassette of recombinase BPR2 | This work |
pKAR3 | pKA, with the expression cassette of recombinase BPR3 | This work |
pKAR4 | pKA, with the expression cassette of recombinase BPR4 | This work |
pKAR5 | pKA, with the expression cassette of recombinase BPR5 | This work |
Primers | ||
amyL-HindIII-F | AAGCTTACGGCTTTATGCCCGATTGC | amyL deletion cassette |
amyL-EcoRI-R | GAATTCCGATCCGCCGTTTACGTGAA | |
Prha-egfp-F | AAAACGCTTTGCCCAAGCTTTCCTGACCCCTCCTTTTAAAAAACATGAG | egfp gene sequence |
Prha-egfp-R | ACCATGGATCCGCGACCCATACGTATCACTCCGTTTTTGTTTGTTT | |
Prha-R-1 | TTTTTTCGTCGCCATACGTATCACTCCGTTTTTGTTTGTTT | BPR1 expression cassette |
BPR1-F | AACGGAGTGATACGTATGGCGACGAAAAAACAAGAAGAAC | |
BPR1-EcoRI-R | GAATTCTTATTCGTTGGTTTCGCCGC | |
Prha-R-2 | CCGGCCCGTATCCATACGTATCACTCCGTTTTTGTTTGTTT | BPR2 expression cassette |
BPR2-F | AACGGAGTGATACGTATGGACACCGGCAGGAAG | |
BPR2-EcoRI-R | GAATTCCTCGTTGGTCTCGCCGC | |
Prha-R-3 | TTTTTTCGTCGTCATACGTATCACTCCGTTTTTGTTTGTTT | BPR3 expression cassette |
BPR3-F | AACGGAGTGATACGTATGACCACCAAGAAGCAGAGC | |
BPR3-EcoRI-R | GAATTCCTCGTTGTCCTTCACCTCCAG | |
Prha-R-4 | TTCGTTTTTCGCCATACGTATCACTCCGTTTTTGTTTGTTT | BPR4 expression cassette |
BPR4-F | AACGGAGTGATACGTATGGCCACCGAGAAGCAG | |
BPR4-EcoRI-R | GAATTCCTGCTCGTCCACCACCTC | |
Prha-R-5 | TTTTTCCGTCGCCATACGTATCACTCCGTTTTTGTTTGTTT | BPR5 expression cassette |
BPR5-F | AACGGAGTGATACGTATGGCCAAGAACGAGGACATCA | |
BPR5-EcoRI-R | GAATTCGTCGAAGGGCAGGTCGTC | |
amyL-YZ-F | CAGAAGCGGCGGAAGAGATT | Diagnostic PCR |
amyL-YZ-R | ACGTTGCCATTTCATCCCCG | |