Table 2.
Strains and plasmid constructs used in this study.
| Strain or plasmid | Genotype and/or characteristics | Source |
|---|---|---|
| E. coli strains | ||
| Mach-1™-T1R | ϕ80lacZΔM15 ΔlacX74 hsdR (rK−, mk+) ΔrecA1498 endA1 tonA | Invitrogen |
| BL21 Star™ (DE3) | F− ompT hsdSB (rB−mB−) gal dcm rne131 (DE3) | Invitrogen |
| BL21 Star™ (DE3) pLysS | F− ompT hsdSB (rB−mB−) gal dcm rne131 (DE3) pLysS (CamR) | Invitrogen |
| Top10 | F− mcrA Δ (mrr-hsdRMS-mcrBC) Φ80lacZΔM15 ΔlacX74 recA1 araD139, Δ (ara leu)7697 galU galK rpsL (StrR) endA1 nupG | Invitrogen |
| B. burgdorferi strains | ||
| ML23 | B. burgdorferi strain B31 clonal isolate missing lp25 | Labandeira-Rey and Skare (2001) |
| ML23 pBBE22luc | Clonal isolate of strain B31 lacking lp25 containing bbe22 and B. burgdorferi codon-optimized luc gene under the control of a strong borrelial promoter (PflaB-luc); parent strain | Hyde et al. (2011a) |
| HZ001 | ML23 Δbba33::StrR | This study |
| HZ001 pBBE22luc | ML23 Δbba33::StrR, containing bbe22 and the B. burgdorferi codon-optimized luc gene under the control of a strong borrelial promoter (PflaB-luc) | This study |
| HZ001 pHZ300 | ML23 Δbba33::StrR, containing bbe22, bba33, both under the control of their native promoters, and the B. burgdorferi codon-optimized luc gene under the control of a strong borrelial promoter (PflaB-luc) | This study |
| Plasmids | ||
| pCR2.1 | pCR™2.1-TOPO® vector; AmpR, KanR | Invitrogen |
| pKFSS1 | B. burgdorferi shuttle vector containing PFlgB-StrR cassette; SpcR in E. coli, StrR in B. burgdorferi | Frank et al. (2003) |
| pBBE22Gate | pBBE22 modified to be a gateway destination vector containing attL and attR sites; CamR, KanR | Weening et al. (2008) |
| pBBE22luc | Borrelial shuttle vector containing bbe22 and B. burgdorferi codon-optimized luc gene under the control of a strong borrelial promoter (PflaB-luc) | Hyde et al. (2011a) |
| pCR2.1Bactin | β-actin gene cloned into pCR2.1 vector; KanR | Hyde et al. (2011a) |
| pCR2.1recA | 1119 bp fragment, containing the recA gene, cloned into pCR2.1 vector; KanR | Hyde et al. (2011a) |
| pET15b | Cloning vector for overexpression of genes to generate His-tagged recombinant protein; AmpR | EMD Millipore |
| pSS008 | bba33 lacking its leader peptide (including the cysteine residue) was cloned into the NdeI and BamHI sites of pET15b in order to produce His-tagged recombinant BBA33 | This study |
| pHZ001 | PCR amplicons containing sequences 1526 bp upstream of the start of B. burgdorferi bba33, the PflgB-StrR cassette from pKFSS1 and sequences 1527 bp downstream of the bba33 stop codon, as well as pCR2.1, were specifically configured using Gibson assembly | This study |
| pHZ300 | Intact bba33 containing sequences 210 bp upstream and 129 bp downstream were cloned into the BamHI and SalI sites of pBBE22luc | This study |