Name | Description | Source |
---|---|---|
Bacterial strains | Â | Â |
EC10B | E. coli DH10B derivative, with repA integrated into the glgB gene. Kanr. | [20] |
NZ9000 | Nisin responsive L. lactis MG1363 derivative, with nisRK integrated into the pepN gene. | [26] |
EGD-e | L. monocytogenes 1/2a strain. Genome sequenced. Obtained from Werner Goebel. | [39] |
EGD-eΔinlA | EGD-e with the E-cadherin interacting region of InlA deleted (amino acids 80 to 506) | [20] |
EGD-eΔinlA::pIMK2inlA | EGD-e ΔinlA with InlA over expressed from the Phelp promoter integrated at tRNAArg locus, Kanr | [20] |
EGD-e InlAm* | EGD-e with inlA residues S192N and Y369 S modified in the chromosome. | This study |
EGD-e A | EGD-eΔinlA with inlA locus recreated containing SDM change N259Y in the chromosome. | This study |
EGD-e B | EGD-eΔinlA with inlA locus recreated containing SDM change Q190L in the chromosome. | This study |
EGD-e C | EGD-eΔinlA with inlA the locus recreated containing SDM changes S173I, L185F and L188F in the chromosome. | This study |
EGD-e D | EGD-eΔinlA with inlA locus recreated containing SDM changes T164A, K301I and G303E in the chromosome. | This study |
EGD-e InlAm* ::pIMC3ery | EGD-e InlAm* with the IPTG inducible expression of erythromycin integrated in the tRNAARG locus, Cmr. | This study |
EGD-e::pIMC3kan | EGD-e with the IPTG inducible expression of kanamycin integrated in the tRNAARG locus, Cmr. | [18] |
EGD-e A::pIMC3kan | EGD-e A with the IPTG inducible expression of kanamycin integrated in the tRNAARG locus, Cmr | This study |
EGD-e B::pIMC3kan | EGD-e B with the IPTG inducible expression of kanamycin integrated in the tRNAARG locus, Cmr | This study |
EGD-e C::pIMC3kan | EGD-e C with the IPTG inducible expression of kanamycin integrated in the tRNAARG locus, Cmr | This study |
EGD-e D::pIMC3kan | EGD-e D with the IPTG inducible expression of kanamycin integrated in the tRNAARG locus, Cmr | This study |
NZ9700 | Nisin producer, progeny of NIZO B8 and MG1363 (Rifr and Strpr) conjugation. | [26] |
Plasmids | Â | Â |
pNZB | Nisin inducible plasmid with heterologous gene expressed from the nisA promoter. Bgl II site upstream of nisA removed. | This study |
pNZBinlAWT | Internalin A from EGD-e containing the entire gene including signal sequence. Cloned into Nco I/Pst I of pNZB. | This study |
pNZBinlAm* | Internalin A containing S192N and Y369 S in pNZB. | This study |
pNZBinlA Bank-iii | Error Prone PCR with low level of mutation 0-4.5 nt per kb. | This study |
pNZBinlA Bank-iv | Error Prone PCR with medium level of mutation 4.5-9 nt per kb. | This study |
pNZBinlA Bank-v | Error Prone PCR with high level of mutation 9-16 nt per kb. | This study |
pNZBinlA Bank-vi | Error Prone PCR with very high level of mutation 9-16 nt per kb. | This study |
pORI280 | RepA negative gene replacement vector, constitutive lacZ, 5.3 kb, Emr. | [40] |
pORI280inlA(SDM) | PCR amplified mutated inlAm* into pORI280 as Nco I/Pst I fragment. Contains wild type inlA promoter. | This study |
pORI280inlA(A) | PCR amplified mutated inlA (from bank v clone 6 containing N259Y) into pORI280 as Nco I/Pst I fragment. Contains Wt inlA promoter. | This study |
pORI280inlA(B) | PCR amplified mutated inlA (from bank iii clone 3 containing Q190L) into pORI280 as Nco I/Pst I fragment. Contains Wt inlA promoter. | This study |
pORI280inlA(C) | PCR amplified mutated inlA (from bank v clone 6 containing S173I, L185F, L188F) into pORI280 as Nco I/Pst I fragment. Contains Wt inlA promoter. | This study |
pORI280inlA(D) | PCR amplified mutated inlA (from bank v clone 8 containing T164A, K301I, G303E) into pORI280 as Nco I/Pst I fragment. Contains Wt inlA promoter. | This study |
pVE6007 | Temperature-sensitive helper plasmid, supplies RepA in trans. Cmr. | [41] |
Name | Oligonucleotide sequence (5'-3') a | Restriction site |
IM194 (inlA-F) | ATATCCATGG AAAAAAACGATATGTATGGTTG | Nco I |
IM188 (inlA-R) | TTTTCTGCAG TTATTTACTAGCACGTGCTTTTTTAG | Pst I |
IM345 (S192N SDM-F) | CAGGTTTAACTAGTCTACAGCAATTAAAT TTTGGTAATCAAGTGACAGATTTAAAACC | Â |
IM346 (S192N SDM-R) | GGTTTTAAATCTGTCACTTGATTACCAAAATTTAATTGCTGTAGACTAGTTAAACCTG | Â |
IM349 (Y369 S SDM-F) | CAAAGCTTCAAAGATTATTTTTCTCT AATAACAAGGTAAGTGACGTAAG | Â |
IM350 (Y369 S SDM-R) | CTTACGTCACTTACCTTGTTATTAGAGAAAAATAATCTTTGAAGCTTTG | Â |
IM490 (Chromosome-F) | ATATCCATGG AAAAGGAGTGTATATAGTG AGAAAAAAACGATATGTATGG | Nco I |
IM466 (Chromosome-R) | ATATCTGCAG CAAACGTTGCTGTATAGCTATTGG | Pst I |
IM467 (inlA out-F) | TATATAGGAAAAATGTGCTGGAACG | Â |
IM468 (inlA out-R) | TCCTTGATAGTCTACTGCTTGAGTCG | Â |
IM317 (inlA muta-F) | AAACAGATCT AGACCAAGTTACAACG | Bgl II |
IM318 (inlA muta-R) | AATTCCA CTTCTTTGG TTGTTTCTTTGC | Bst XI |