Table 3.
Details of oligonucleotides used in this study. The restriction sites within the oligonucleotide sequences are mentioned in italics.
| Sl. no. | Primer name | Sequences (5′ → 3′) | Target gene and purpose of use |
|---|---|---|---|
| 1. | NptII-F | TAGACTGGGCGGTTTTATGGACAG | nptII (encodes neomycin phosphotransferase II enzyme that metabolizes neomycin and kanamycin and gives resistance against these antibiotics to the organism harboring this gene). Used to detect the presence nptII gene in the transformants produced after transposon mediated mutagenesis |
| NptII-R | AACTCCGCGAGGTCGTCCAGCCTC | ||
| 2. | MproC-F | CTTCAAGCTTCCAGCTTGTCCTATCTGTTCTT | proC (encodes pyrroline-5-carboxylate reductase, one of the genes involved in proline synthesis). Used to amplify the internal fragment of proC and its subsequent cloning into pMUTIN4 for targeted inactivation of proC |
| MproC-R | CGTTGGATCCTCAGGATCGAGACCTTCTTCT | ||
| 3. | ermAM-F | GAACAAAAATATAAAATATTCTCG | ermAM (encodes the enzyme Adenine methylase that metabolize erythromycin and gives resistance against these antibiotics to the organism harboring this gene) Used to detect the presence ermAM gene in the transformants produced after transformation of pMutproCi |
| ermAM-R | TCCTCCCGTTAAATAATAGATAACT | ||
| 4. | RT16S-F | GTGTCGTGAGATGTTGGGTTA | 16S rRNA gene (used as endogenous control in qRT-PCR) |
| RT16S-R | GTGTGTAGCCCAGGTCATAAG | ||
| 5. | RTproC-F | GGAAGTGGACCCGCTTATTT | proC (used to measure the differential expression of proC through qRT-PCR |
| RTproC-R | TCGGCGTTTCATCTCTTTCC | ||
| 6. | Exp-proC-F | CCTCTAGAATGGCAGAAGCGATGATTTCTG | proC (used to amplify the internal fragment of proC gene for cloning into pHT01 plasmid and expression analysis) |
| Exp-proC-R | CGCCCGGGTTAATTAGTACTTGCCACTTTTTGTAG |