TABLE 2.
E. coli strains and plasmids used in this study
| Strain or plasmid | Genotype and/or characteristicsa | Reference or source |
|---|---|---|
| E. coli strains | ||
| DH5α | F−
endA1 hsdR17 supE44 thi-1 recA1 gyrA96 relA1 Δ(argF-lacZYA) U169 (Φ60ΔlacZ M15λ−) |
77 |
| Rosetta (DE3) |
E. coli strain B; F−
ompT gal dcm lon? hsdSB(rB−mB−) λ(DE3 [lacI lacUV5-T7p07 ind1 sam7 nin5]) [malB+]K-12(λS) |
Novagen |
| One Shot pSLC-242 | Fc
mcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 Δ lacX74 recA1 araD139 Δ(araleu)7697 galU galK rpsL (Smr) endA1 nupG |
AddGene/ ThermoFisher |
| SY327λpir | λ(lac pro) argE (Am) rif nalA recA56 (λpir) | 78 |
| MG1655 | K-12 F− λ ilvG rfb-50 rph-1 | 79 |
| M1/5 | Fecal isolate of a healthy individual; pks+, HPI+ | This study |
| Nissle 1917 | Fecal isolate of a healthy individual; pks+, HPI+ | 80 |
| IHE3034 | Newborn meningitis E. coli isolate; pks+, HPI+ | 81 |
| SP15 | Newborn meningitis E. coli isolate; pks+, HPI+ | 82 |
| UTI89 | Uropathogenic E. coli isolate; pks+, HPI+ | 83 |
| Nissle 1917 λ-attB::5VNTR- clbRp-lux |
Nissle 1917 clbR promoter region containing 5 VNTRs fused with luxABCDE integrated into the λ-attB site |
This study |
| IHE3034 λ-attB::5VNTR- clbRp-lux |
IHE3034 clbR promoter region containing 5 VNTRs fused with luxABCDE integrated into the λ-attB site |
This study |
| SP15 λ-attB::5VNTR-clbRp-lux | SP15 clbR promoter region containing 5 VNTRs fused with luxABCDE integrated into the λ-attB site |
This study |
| UTI89 λ-attB::5VNTR-clbRp-lux | UTI89 clbR promoter region containing 5 VNTRs fused with luxABCDE integrated into the λ-attB site |
This study |
| M1/5 rpsLK42R | M1/5 carrying a rpsLK42R mutation; Smr | 51 |
| M1/5 rpsLK42R 19VNTR | M1/5 rpsLK42R VNTR region adjusted via scarless mutagenesis to 19 repeats | This study |
| M1/5 rpsLK42R 5VNTR | M1/5 rpsLK42R VNTR region adjusted via scarless mutagenesis to 5 repeats | This study |
| M1/5 rpsLK42R ΔclbQ | M1/5 rpsLK42R ΔclbQ::FRT | This study |
| M1/5 rpsLK42R ΔclbR | M1/5 rpsLK42R ΔclbRA::clbA-FRT | This study |
| M1/5 rpsLK42R λ-attB::FRT | FRT site integrated at λ-attB site | This study |
| M1/5 λ-attB::20VNTR-clbRp-lux | M1/5 clbR promoter region containing 20 VNTRs fused with luxABCDE integrated into the λ-attB site |
This study |
| M1/5 λ-attB::5VNTR-clbRp-lux | M1/5 clbR promoter region containing 5 VNTRs fused with luxABCDE integrated into the λ-attB site |
This study |
| Plasmids | ||
| pASK75 | Template for tetA promoter cloning | 84 |
| pBAD24 | Ampr; araC; araBADp | 85 |
| pBAD-clbR | For l-arabinose-inducible expression of clbR from pBAD24 | This study |
| pBAD24-clbQ-rrnBt | Template for cloning | This study |
| pBAD24-tetAp-clbQ-rrnBt | For constitutive expression of clbQ | This study |
| pBAD24-tetAp-clbR-rrnBt | For constitutive expression of clbR | This study |
| pBR322 | Template for rrnB terminator cloning | 86 |
| pCP20 | Temperature-sensitive origin of replication, encodes Flp recombinase; Ampr, Cmr | 73 |
| pEX-K4-tetAp-clbR-rrnBt | Contains synthetic tetAp-clbR-rrnBt insert for clbR expression | This study |
| pFuseA-npt | pGP704 derivative for chromosomal integration via a FRT sequence; Kanr; oriR6K; luxABCDE |
33 |
| pFuseA-npt-20VNTR-clbRp-lux | pFuseA-npt derivative carrying 20 VNTRs upstream of a clbR promoter-luxABCDE fusion; Kanr; oriR6K |
This study |
| pFuseA-npt-5VNTR-clbRp-lux | pFuseA-npt derivative carrying 5 VNTRs upstream of a clbR promoter-luxABCDE fusion; Kanr; oriR6K |
This study |
| pGEM-T Easy | TA cloning vector | Promega |
| pGEM-T Easy-tetAp-clbR | pGEM-T Easy derivative carrying a tetAp-clbR-rrnBt fusion for inducible expression of clbR; Ampr |
This study |
| pKD3 | Template plasmid for amplification of the FRT-flanked chloramphenicol resistance cassette; FRT-cat-FRT; Ampr, Cmr |
71 |
| pKD3-ΔclbR1 | pKD3 derivative in which the FRT site upstream of cat has been replaced by a sequence containing the clbR upstream region and the clbA gene; clbRp::clbA- cat-FRT; Ampr, Cmr |
This study |
| pKD4 | Template plasmid for amplification of the FRT-flanked kanamycin resistance cassette; Ampr, Kanr |
71 |
| pKD4-'clbA | pKD3 derivative with an insertion of the last 524 bp of the clbA gene upstream of the FRT-flanked npt cassette; 'clbA-FRT-npt-FRT; Ampr, Kanr |
This study |
| pKD46 | Helper plasmid for l-arabinose-inducible expression of λ-Red recombinase (araC araBp- γ-β-exo); Ampr |
71 |
| pSLC-242 | Template plasmid cat cassette for positive selection and relE toxin gene under the control of rhamnose inducible promoter (rhaBp) for negative selection; Cmr |
72 |
| pTXB1_clbR | pTXB1 with clbR gene of E. coli Nissle 1917, for IPTG-inducible expression of a ClbR intein/chitin binding domain fusion for purification; Ampr |
This study |
| pUC57-Insert_pFuseA-npt_ clbR_20VNTR |
Contains synthetic insert with 20-repeat VNTR-clbRp-lux fusion | This study |
a
Ampr, ampicillin resistance; Cmr, chloramphenicol resistance; IPTG, isopropyl-β-d-thiogalactopyranoside; Kanr, kanamycin resistance; Smr, streptomycin resistance; HPI, high-pathogenicity island.