TABLE 1.
Primers and probes
| Primers or probes | Nucleotide sequence (5′–3′)a | Product or target |
|---|---|---|
| Primers | ||
| ermKpnIF | aaGGTACCCCCGATAGCTTC | ermFermAM cassette |
| ermSphIR | attGCATGCCGAAGCTGTCAGTAGTATACC | ermFermAM cassette |
| tetQKpnIF | tcGGTACCAACCCCGTTATATACATTCATGT | tetQ cassette |
| tetQSphIR | actGCATGCCAAGTTCTAATGCTTCTATCTC | tetQ cassette |
| dpsfrASacIF | aaGAGCTCATAGAACTGGAATCTGCCAAG | pWDdps construction |
| dpsfrAKpnIR | caGGTACCAATCTTCTACGTTTTTAATTGTGTTCC | pWDdps construction |
| dpsfrBSphIF | atGCATGCTACGCTGCCAAGTAAAAAC | pWDdps and pWCdps construction |
| dpsfrBHindIIIR | ccAAGCTTCTCCGAACATCTCAATACC | pWDdps and pWCdps construction |
| dpsfrCEcoRIF | gtGAATTCGGACTTCTTCTGGAAAGCA | pWCdps construction |
| dpsfrCSacIR | ttGAGCTCTCACTCTTGGGTTTCTGC | pWCdps construction |
| dpsFrCF | CGATACGGGAGTCTTCAATATC | Crossover confirmation PCR |
| dpsFrBR | ATACCTGCGAAGGGTAGAT | Crossover confirmation PCR |
| dpsF | AAAAGATTCTTGAAGTAACGGGTT | Sequencing primer |
| dpsR | TCCAAACGAGCTTCTCTTG | Sequencing primer |
| DpsCteminus XhoIR | aCTCGAGTTATTTTTCAAACTGCGGATGGCTCCACGGGCCCTGAAACAGCACTTCCAGCTTGGCAGCGTAGGCAGA | pETdps construction |
| DpsNcoIF | agaCCATGGgtATGAAAAAGATTCTTGAAGTAACG | pETdps construction |
| DpsC101AFA | GAACTCGTTGCGGCTGCAAGTACGCTGAAG | pDpsC101A mutagenesis |
| DpsC101AFB | AGTACGCTGAAGAATGTGAC | pDpsC101A mutagenesis |
| DpsC101ARA | TGCAGCCGCAACGAGTTCGTGCTCTTC | pDpsC101A mutagenesis |
| DpsC101ARB | GTGCTCTTCCTTCACTTCTG | pDpsC101A mutagenesis |
| RTdpsF | GTGAAGGAAGAGCACGAACTCG | qPCR of dps |
| RTdpsR | CTGCCTCACCGGCAACTTC | qPCR of dps |
| RT16sF | TCGGTAAGTCAGCGGTGAAAC | qPCR of 16S RNA |
| RT16sR | ATTGCGAAGGCAGCTTGC | qPCR of 16S RNA |
| Fluorescent-quencher probes | ||
| FAMRTdpsPb | CGCTGCAGATCATCATGGCGAAGGAGCGTGC | qPCR probe for dps |
| CY5RT16sPc | AGCGCTCAACGTTCAGCCTGCCGTTGA | qPCR probe for 16S RNA |
a Restriction sites are in bold; PrescissionTM cleavage site is in capital italics; Strep-tag II is underlined, and irrelevant bases are in lowercase.
b 5′-Fluorescent dye is 6-carboxyfluorescein (FAM), and 3′-quencher dye is IowaBlackTM FQ (Integrated DNA Technologies Inc., Coralville, IA).
c 5′-Fluorescent dye is Cy5TM, and 3′-quencher dye is IowaBlackTM RQ (Integrated DNA Technologies Inc.).