Table 1.
Examples of primers used to identify and confirm the presence of the insertion of interest (Table 1). Primers were designed based on sequences of the transposon (encoding mCherry or aadA) and flanking genome regions identified by NextGen sequencing (Illumina HighSeq). Primer pairs to facilitate PCR amplification of DNA spanning the 5’ or 3’ transposon termini and adjacent genome regions were used to identify specific mutants in samples pooled for Illumina sequencing. Primer pairs complementary to genomic regions flanking the transposon were used to verify that a transposon was present at the target site.
| Primer List for selected mutants | Pairs with | |
|---|---|---|
| Spec out | GTATCAGCCCGTCATACTTGAA | - |
| Cherry out | GACCTTAAGTTTAGCCGTCTGT | - |
| ChUp&Out | ATTATCTTCCTCTCCCTTGCTGACC | - |
| 00035 at 7443 (isoprenoid biosynthesis protein) | ACTAAGCACTCATGACGTAAGGTCT | ChUp& out |
| 00035 at 7443 ID | ACACTACTAAGAGAAGCAGCAACGA | 00 035 at 7443 |
| HGE1 00140 FOR (hypothetical protein) | TGACGTTACCGTGCTCGAAG | Cherry out |
| HGE1 00140 REV | GTAATCCAGTCCCTGCCGAG | Spec out |
| HGE1 00255 FOR (hypothetical protein) | GAGCATGAGTCCGTGGGTAG | Spec out |
| HGE1 00255 REV | ATATGCTGAAACGTGCGCTG | Cherry out |
| HGE1 01752 FOR (HGE-14 protein) | ATGCCGTGGGTTCTTACGAG | Spec out |
| HGE1 01752 REV | GCATCCAAGCATAGCTGCAC | Cherry out |
| HGE1 05312 FOR (DNA-binding protein) | TGCGCAACAACTTTAAGCCC | Cherry out |
| HGE1 05312 REV | AGCGTAGAATAGGCGAACCG | Spec out |
| HGE1 05592 FOR (MerR family transcriptional regulator) | AAAAAGGAGAGGAAGGCGCT | Spec out |
| HGE1 05592 REV | GTCTCAACAGAGTTGCGCTG | Cherry out |