TABLE 3.
Primers used in this work
| Function and name | Sequence (5′ → 3′)a | PCR template |
|---|---|---|
| tra trb assembly | ||
| FtraHind | CATACCCTTTCGAAGCTTTCAGC | pCTX-M3 |
| RtraPst | CTCCTGCTGCAGTTTCTGTGC | pCTX-M3 |
| FnicM | GTACGGGACAATATTGGTTTTTGGAGTACCGC | pCTX-M3 |
| RnicM | CTCCAAAAACCAATATTGTCCCGTACTTAAATACC | pCTX-M3 |
| FtraSal | GCAGGGTCGACTTCTATCTTCGCTAGCGG | pCTX-M3 |
| RtraXba | ACTCTCTCTAGAACTCCGGGTTAC | pCTX-M3 |
| FtrbXba | AGATCTAGAAAACGTTGCTTAACGTGAG | pCTX-M3 orf46::cat |
| RtrbBam | TTCCAGGATCCCCTGGTACGCAGCGCAG | pCTX-M3 orf46::cat |
| FtrbNco-Sac | CGGTTGAGCTCGTCGAGAATGGATTTAGC | pCTX-M3 |
| RtrbEco | AATAGAATTCCTCTGACACCCTCTC | pCTX-M3 |
| FrepCNI | GTGGCGGCCGCGTAAGAAACCATTATTATC | pBS3-1 |
| RrepANB2 | TAGGCGGCCGCGGTCTCGCACCCCTGCCGTCTTACG | pBS3-1 |
| Nick region mutagenesis | ||
| FAatII | TTCTGACGTCACATCAGGCAAGTCG | pLMAB212 |
| RnicSpe | AACCGAACTAGTCCCGTACTTAAATACCTC | pLMAB212 |
| FKanSpe2 | GAACTAGTCATGAACAATAAAACTGTCTGC | pET28a+ |
| RKanSpe | AGACTAGTATCCGCTCATGAATTAATTC | pET28a+ |
| FnicSpe | GGACTAGTTCGGTTTTTGGAGTACCGCCGACAC | pLMAB212 |
| RPshAI | GAAGACCGATGTCTGCAAATGTCTTATGC | pLMAB212 |
| Kan-oriT cloning | ||
| FKanAatII | ATGGACGTCAGCTACTGGGCTATCTGG | pToriTB |
| oriTminDAatII | TTGGACGTCTGCAGAGATAGCTAACCTCGTTAGG | pToriTB |
| orf36 replacement with cat | ||
| orf36uP1 | ATGCAAACAGTGATGCATTCCCGTTCCATTTGTAACGTGTAGGCTGGAGCTGCTTCG | pKD3 |
| orf36dP2 | GAACAATGAGGTATACATGAGCGAACATAATGATTATATATGAATATCCTCCTTA | pKD3 |
| Integration verification | ||
| ybhB122 | CTGGCAAGCGCCTCGATTAC | |
| ybhC159 | ACCAGGCGCGGTTTGATCAG | |
| orf35UEc | TCGAATTCGACATTATTGGGAGGGC | |
| FtrbNco-Sac | CGGTTGAGCTCGTCGAGAATGGATTTAGC | |
| pCTX96 | CCGAGTCAGTTTGATCCATA | |
| orf36sU | GGATGAGGTATGCAATACGG | |
| Cloning of zeta gene | ||
| EcoZetaFor | GCC GAA TTC ATG GCA AAT ATA GTC AAT TTT ACT | pBT233 |
| ZetaRevBam | GCC GGA TCC TTA AAT ACC TGG AAG TTT AGG TGT | |
| Cloning of PBAD promoter | ||
| upTEM | CACCAGCGTTTCTGGGTGAG | pBAD24 |
| ARA1down | GCTCTAGAGGCGTCACACTTTGCTATGC | |
| epsilon gene detection | ||
| EpsiS | TGAAATGGCAGTTACGTATG | |
| EpsiE | TGCCATATTAAGCCACTTTC |
a
Relevant restriction sites are underlined. epsilon gene start and stop codons are in bold.