Table 4.
Oligonucleotides used in this study.
| Purpose | Oligonucleotide pair(s) |
|---|---|
| iglE null mutant | |
| IglE Δ6–119 | FTL0124_a: 5´-CTC GAG ATA ATA TCG CTA GCT AAA AGA C-3´ (XhoI) + FTL0124_b3: 5´-GGA TCC AGT AGC ATA GAA AAA GAT TAA GG-3´ (BamHI) |
| FTL0124_c2: 5´-GGA TCC TAA TTT ATT GTA CAT TGA CTT CTC-3´ (BamHI) + FTL0124_d: 5´-GAG CTC TGA TAG CCT TAA TAA TGA CTC T-3´ (SacI) | |
| Expression in trans | |
| IglE | IglE_NdeI_F2: 5´-CT ATG TAC AAT AAA TTA TTG AAA AAT CTT TGT TTA GTA-3´ (NdeI) + PigA_GSK_rev: 5´-GGT ACC ATC TTT TTC TAT GCT ACT ATC-3´ (KpnI) |
| IglE Δ2–22 | IglE_D2–22_F: 5´-CT ATG ATT AGT GAT GGT TTG TAT ATC AAC-3´ (NdeI) + PigA_GSK_rev (KpnI) |
| IglE Δ23–43 | IglE_NdeI_F2 (NdeI) + IglE_D23–43_b: 5´-ACA ACT GCT AAG CCC TAT GAT-3´ |
| IglE_D23–43_c: 5´-AGG GCT TAG CAG TTG TCC TGA TAA AAA TAT TTT CTA CTC AG-3´ + PigA_GSK_rev (KpnI) | |
| IglE Δ44–64 | IglE_NdeI_F2 (NdeI) + IglE_D44–64_b: 5´-TTT AGA TTC TAG AAC TAT TTT TGT C-3´ |
| IglE_D44–64_c: 5´-AGT TCT AGA ATC TAA AAA TGT AAA AGT ATT AAA TCT TAA AAC A-3´ + PigA_GSK_rev (KpnI) | |
| IglE Δ65–85 | IglE_NdeI_F2 (NdeI) + IglE_D65–85_b: 5´-ATC ATC ATA TAT TCT TTG AGA AAT A-3´ |
| IglE_D65–85_c: 5´-AAG AAT ATA TGA TGA TGA TTA TGC CTT GTA TTT TAT ACT TC-3´ + PigA_GSK_rev (KpnI) | |
| IglE Δ86–105 | IglE_NdeI_F2 (NdeI) + IglE_D86–105_b: 5´-CTT GAT ATC TTT ATC TAA TGG AA-3´ |
| IglE_D86–105_c: 5´-AGA TAA AGA TAT CAA GAT AAG TTC AGA TTC TGT AAA TAA ATT-3´ + PigA_GSK_rev (KpnI) | |
| IglE Δ106–125 | IglE_NdeI_F2 (NdeI) + IglE_D106–125_KpnI: 5´-GGT ACC TAT GTA TTT CCA GTT TTC AGT TT-3´ (KpnI) |
| IglE L19A | IglE_NdeI_F2 (NdeI) + IglE_L19A_b: 5´-CCC CTA TGA TTG TGG ATA ATA CT-3´ |
| IglE_L19A_c: 5´-TCC ACA ATC ATA GGG GCT AGC AGT TGT ATT AGT GAT G-3´ + PigA_GSK_rev (KpnI) | |
| IglE S20A | IglE_NdeI_F2 (NdeI) + IglE_S20A_b: 5´-CAA GCC CTA TGA TTG TGG ATA ATA-3´ |
| IglE_S20A_c: 5´-CAA TCA TAG GGC TTG CTA GTT GTA TTA GTG ATG GTT TG-3´ + PigA_GSK_rev (KpnI) | |
| IglE S21A | IglE_NdeI_F2 (NdeI) + IglE_S21_b: 5´-CGC TAA GCC CTA TGA TTG TGG ATA ATA-3´ |
| IglA_S21A_c: 5´-ATC ATA GGG CTT AGC GCT TGT ATT AGT GAT GGT TTG-3´ + PigA_GSK_rev (KpnI) | |
| IglE C22A | IglE_NdeI_F2 (NdeI) + IglE_C22A_b: 5´-CAC TGC TAA GCC CTA TGA TTG T-3´ |
| IglE_C22A_c: 5´-TAG GGC TTA GCA GTG CTA TTA GTG ATG GTT TGT ATA T-3´ + PigA_GSK_rev (KpnI) | |
| IglE I33F | IglE_NdeI_F2 (NdeI) + IglE_33_b: 5´-CTA TTT TTG TCT TAG GGA AAT TGT TGT TGA TAT ACA AAC CT C-3´ |
| IglE_33_c: 5´-TTT CCC TAA GAC AAA AAT AGT TCT AGA ATC-3´ + PigA_GSK_rev (KpnI) | |
| IglE P34L | IglE_NdeI_F2 (NdeI) + IglE_34_b: CTA TTT TTG TCT TTA GAA TAT TGT TGT TGA TAT ACA AAC CT-3´ |
| IglE_34_c: 5´-TCT AAA GAC AAA AAT AGT TCT AGA ATC TAA-3´ + PigA_GSK_rev (KpnI) | |
| IglE K35R | IglE_NdeI_F2 (NdeI) + IglE_35_b: 5´-ACT ATT TTT GTT CTA GGA ATA TTG TTG TTG ATA TAC AAA C-3´ |
| IglE_35_c: 5´-TAG AAC AAA AAT AGT TCT AGA ATC TAA AC-3´ + PigA_GSK_rev (KpnI) | |
| IglE T36Q | IglE_NdeI_F2 (NdeI) + IglE_36_b: 5´-AGA ACT ATT TTT TGC TTA GGA ATA TTG TTG TTG ATA TAC A-3´ |
| IglE_36_c: 5´-GCA AAA AAT AGT TCT AGA ATC TAA ACC TG-3´ + PigA_GSK_rev (KpnI) | |
| IglE I38Y | IglE_NdeI_F2 (NdeI) + IglE_38_b: 5´-ATT CTA GAA CT ATT TTG TCT TAG GAA TAT TGT TGT TG-3´ |
| IglE_38_c: 5´-AAT ATG TTC TAG AAT CTA AAC CTG ATA AA-3´ + PigA_GSK_rev (KpnI) | |
| IglE I33F, I38Y | IglE_NdeI_F2 (NdeI) + IglE_33_38_b: 5´-ATA TTT TGT CTT AGG GAA ATT GTT GTT GAT ATA CAA ACC AT-3´ |
| IglE_33_38_c: 5´-TTT CCC TAA GAC AAA ATA TGT TCT AGA ATC-3´ + PigA_GSK_rev (KpnI) | |
| IglE P34L, I38Y | IglE_NdeI_F2 (NdeI) + IglE_34_38_b: 5´-ATA TTT TGT CTT TAG AAT ATT GTT GTT GAT ATA CAA ACC AT-3´ |
| IglE_34_38_c: 5´-TCT AAA GAC AAA ATA TGT TCT AGA ATC-3´ + PigA_GSK_rev (KpnI) | |
| IglE K35R, I38Y | IglE_NdeI_F2 (NdeI) + IglE_35_38_b: 5´-ACA TAT TTT GTT CTA GGA ATA TTG TTG TTG ATA TAC AAA C-3´ |
| IglE_35_38_c: 5´-TAG AAC AAA ATA TGT TCT AGA ATC TAA AC-3´ + PigA_GSK_rev (KpnI) | |
| IglE T36Q, I38Y | IglE_NdeI_F2 (NdeI) + IglE_36_38_b: 5´-AGA ACT ATT TTT TGC TTA GGA ATA TTG TTG TTG ATA TAC A-3´ |
| IglE_36_38_c: 5´-GCA AAA ATA TGT TCT AGA ATC TAA ACC TG-3´ + PigA_GSK_rev (KpnI) | |
| IglE FS1(2–6) | IglE_2–6_F: 5´-CT ATG ACA ATA AAT TAT GTG AAA AAT CTT TGT TTA GTA TT-3´ (NdeI) + PigA_GSK_rev (KpnI) |
| IglE FS2(7–17) | IglE_7–17_F: 5´-CT ATG TAC AAT AAA TTA TTG CAA AAA TCT TTG TTT AGT ATT ATC CAC AAT CT AGG CTT AGC AGT TGT ATT AGT G-3´ (NdeI) + PigA_GSK_rev (KpnI) |
| IglE FS3(23–32) | IglE_NdeI_F2 (NdeI) + IglE_23–32_b: 5´-TGT TGA TAT ACA AAC CT CAC TAA ACA ACT GCT AAG CCC T-3´ |
| IglE_23–32_c: 5´-GGT TTG TAT ATC AAC AAC AAG TAT TCC TAA GAC AAA AAT AGT TC-3´ + PigA_GSK_rev (KpnI) | |
| IglE FS4(33–38) | IglE_NdeI_F2 (NdeI) + IglE_33–38_b: 5´-TAT TTT TGT CTT AGG AAA TTG TTG TTG ATA TAC AAA CCA TC-3´ |
| IglE_33–38_c: 5´-TCC TAA GAC AAA AAT ATG TTC TAG AAT CTA AAC CTG ATA-3´ + PigA_GSK_rev (KpnI) | |
| IglE FS5(2) | IglE_2_F: 5´-CT ATG ACA AAT AAA TTA TTG AAA AAT CTT TGT TTA GTA-3´ (NdeI) + PigA_GSK_rev (KpnI) |
| IglE FS6(2–3) | IglE_2–3_F: 5´-CT ATG ACA ATA AAA TTA TTG AAA AAT CTT TGT TTA GTA TT-3´ (NdeI) + PigA_GSK_rev (KpnI) |
| IglE FS7(2–4) | IglE_2–4_F: 5´-CT ATG ACA ATA AAT TTA TTG AAA AAT CTT TGT TTA GTA TT-3´ (NdeI) + PigA_GSK_rev (KpnI) |
| IglE FS8(2–5) | IglE_2–5_F: 5´-CT ATG ACA ATA AAT TAT TTG AAA AAT CTT TGT TTA GTA TT-3´ (NdeI) + PigA_GSK_rev (KpnI) |
| IglE FS9(3–5) | IglE_3–5_F: 5´-CT ATG TAC ATA AAT TAT TTG AAA AAT CTT TGT TTA GTA TT-3´ (NdeI) + PigA_GSK_rev (KpnI) |
| IglE FS10(3–6) | IglE_3–6_F: 5´-CT ATG TAC ATA AAT TAT GTG AAA AAT CTT TGT TTA GTA TT-3´ (NdeI) + PigA_GSK_rev (KpnI) |
| IglE FS11(4–6) | IglE_4–6_F: 5´-CT ATG TAC AAT AAT TAT GTG AAA AAT CTT TGT TTA GTA TT-3´ (NdeI) + PigA_GSK_rev (KpnI) |
| IglE FS12(5–6) | IglE_5–6_F: 5´-CT ATG TAC AAT AAA TAT GTG AAA AAT CTT TGT TTA GTA TT-3´ (NdeI) + PigA_GSK_rev (KpnI) |
| IglE FS13(6) | IglE_6_F: 5´-CT ATG TAC AAT AAA TTA GTG AAA AAT CTT TGT TTA GTA TT-3´ (NdeI) + PigA_GSK_rev (KpnI) |
| B2H analysis | |
| PdpB Δ2–589 | PdpB_590–1093_F: 5´-CT ATG AGT GTT GTC AAC CCT ACT TAT A-3´ (NdeI) + PdpB_NotI_R: 5´-GGC GCC GC TTG TAC ATT GAC TTC TCC TT-3´ (NotI) |
| IglE Δ2–22 | IglE_D2–22_F (NdeI) + IglE_NotI_Rev: 5´-GCG GCC GC ATC TTT TTC TAT GCT ACT ATC A-3´ (NotI) |
| IglE C22A | IglE_NdeI_F2 (NdeI) + IglE_C22A_b: 5´-CAC TGC TAA GCC CTA TGA TTG T-3´ |
| IglE_C22A_c: 5´-TAG GGC TTA GCA GTG CTA TTA GTG ATG GTT TGT ATA T-3´ + PigA_GSK_rev (KpnI) | |
| IglE C22S | IglE_NdeI_F2 (NdeI) + IglE_C22S_b: 5´-TAC TGC TAA GCC CTA TGA TTG T-3´ |
| IglE_C22S_c: 5´-TAG GGC TTA GCA GTA GTA TTA GTG ATG GTT TGT ATA T-3´ + PigA_GSK_rev (KpnI) | |
| IglE C22G | IglE_NdeI_F2 (NdeI) + IglE_C22G_b: 5´- CAC TGC TAA GCC CTA TGA TTG TGG AT-3´ |
| IglE_C22G_c: 5´-TAG GGC TTA GCA GTG GTA TTA GTG ATG GTT TGT ATA T-3´ + PigA_GSK_rev (KpnI) |
Note. The nucleotide sequences in italics represent the incorporated NdeI, KpnI, XhoI, BamHI, NotI and SacI restriction sites used for cloning of the PCR amplified DNA fragments. Underlined sequences indicate complementary sequences in the overlap PCR primers.