Title: Slam is an outer membrane protein that is required for the surface display of lipidated virulence factors in Neisseria Authors: Yogesh Hooda, Christine ChiehLin Lai, Andrew Judd, Carolyn M. Buckwalter, Hyejin Esther Shin, Scott D. Gray Owen, and Trevor F. Moraes To whom correspondence should be addressed E-mail: [email protected]This PDF file includes: Supplementary Figures 1-12 Supplementary Table 1: Strains, plasmids and antibodies used in this study. SUPPLEMENTARY INFORMATION ARTICLE NUMBER: 16009 | DOI: 10.1038/NMICROBIOL.2016.9 NATURE MICROBIOLOGY | www.nature.com/naturemicrobiology 1
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Material and Methods Supp-Jan14 - Nature Research · 2016-03-29 · pGCC4 Slam1Ntd pGCC4 containing Slam1 N-terminal domain This study pGCC4 TbpB pGCC4 containing full length TbpB
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Title: Slam is an outer membrane protein that is required for the surface display of lipidated virulence factors in Neisseria
Authors: Yogesh Hooda, Christine Chieh-‐Lin Lai, Andrew Judd, Carolyn M. Buckwalter, Hyejin Esther Shin, Scott D. Gray Owen, and Trevor F. Moraes
To whom correspondence should be addressed E-mail: [email protected]
This PDF file includes: Supplementary Figures 1-12 Supplementary Table 1: Strains, plasmids and antibodies used in this study.
Supplementary Table 1: Strains, plasmids and antibodies used in this study. Strain or plasmid Description Reference Strains: N. meningitidis B16B6 [1] N. meningitidis B16B6 nmb0313/tn5
EZTn5-kanamycin insertion in nmb0313 This study
N. meningitidis B16B6 Δnmb0313
Δnmb0313::kan This study
N. meningitidis B16B6 ΔtbpB ΔtbpB::kan [2]
N. meningitidis B16B6 ΔlbpB ΔlbpB::kan [3]
N. meningitidis MC58 [4] N. meningitidis MC58 Δnmb0313
Δnmb0313::kan This study
E. coli MM294 F-, glnX44(AS), LAM-, rfbC1, endA1, spoT1, thiE1, hsdR17, creC510 Yale CGSC #6315 E. coli C43 F– ompT gal dcm hsdSB(rB- mB-)(DE3) [5] E. coli BW25113 F-, Δ(araD-araB)567, ΔlacZ4787(::rrnB-3), λ-, rph-1, Δ(rhaD-rhaB)568, hsdR514 [6] E. coli JW4179-1 F-, Δ(araD-araB)567, ΔlacZ4787(::rrnB-3), λ-, rph-1, Δ(rhaD-rhaB)568,ΔytfM769::kan, hsdR514 [6] E. coli JW4180-1 F-, Δ(araD-araB)567, ΔlacZ4787(::rrnB-3), λ-, rph-1, Δ(rhaD-rhaB)568,ΔytfN770::kan, hsdR514 [6] E. coli E11 K1 capsule type, clinical isolate [7] E. coli E11 ΔpgaB ΔpgaB [7] Plasmids: pUC19 Slam1-Kan pUC19 containing the 500bp upstream and downstream regions of slam1 flanking kan2 from the
EZ::Tn5 transposon kit This study
pGCC4 Neisseria complementation vector [8] pGCC4 Slam1 pGCC4 containing full length Slam1 This study
pGCC4 Slam1barrel pGCC4 containing Slam1 barrel domain This study
pGCC4 Slam1Ntd pGCC4 containing Slam1 N-terminal domain This study pGCC4 TbpB pGCC4 containing full length TbpB This study
pGCC4 GST-C-lobe pGCC4 plasmid with GST inserted after the anchoring peptide, replacing the TbpB N-lobe and in frame with the TbpB C-lobe
This study
DOI: 10.1038/NMICROBIOL.2016.9 SUPPLEMENTARY INFORMATION
pGCC4 GST pGCC4 plasmid with GST inserted after the anchor peptide, replacing both the TbpB N- and C-lobes
This study
pET26b E.coli expression vector Addgene, 69862-3
pET26 Slam1 pET26 containing pelB signal peptide, 7xHis and mature B16B6 Slam1 This study pET26 Slam1barrel pET26 containing pelB signal peptide, 7xHis and B16B6 Slam1 barrel domain This study
pET26 Slam2 pET26 containing pelB signal peptide, 7xHis and mature MC58 Slam2 This study
pETDuet E.coli expression vector Addgene, 71146
pETDuet Slam1 pETDUET containing pelB signal peptide and mature B16B6 Slam1 in MCS site 1 This study pETDuet TbpB pETDUET containing pelB signal peptide and mature B16B6 TbpB in MCS site 2 This study
pETDuet T5 TbpB pETDUET containing full length B16B6 TbpB in MCS site 2 with pT5-Lac promoter This study
pETDuet LolA R43L_TbpB pETDUET containing full length B16B6 TbpB in MCS site 2 and His-LolA R43L in MCS site 1 This study pETDuet LbpB pETDUET containing pelB signal peptide and mature MC58 LbpB in MCS site 2 This study
pETDuet fHbp pETDUET containing pelB signal peptide and mature MC58 fHbp in MCS site 2 This study
pETDuet HpuA pETDUET containing pelB signal peptide and mature B16B6 HpuA in MCS site 2 This study pETDuet GST-C-lobe pETDUET containing full length B16B6 TbpB with the N-lobe replaced by a GST domain, in MCS
site 2 This study
pHERD30T pBAD-based shuttle vector [9]
pHERD30T pelB Slam1 pHERD30T containing pelB signal peptide and mature Slam1, sub-cloned from pET26b This study
α-LepB Rabbit polyclonal antibodies were obtained from Dr. Jan Willem de Gier [11] α -OmpA Rabbit polyclonal antibodies were obtained from Dr. Jan Willem de Gier [12] α-Slam1 Supplementary Figure 10c, rabbit polyclonal sera This study
α-fHbp Mouse monoclonal Jar5 antibodies were obtained from Dr. D. Granoff [13]
α-HpuA Supplementary Figure 10b, rabbit polyclonal sera This study α-TbpB Supplementary Figure 10a, rabbit polyclonal sera This study
DOI: 10.1038/NMICROBIOL.2016.9 SUPPLEMENTARY INFORMATION
α-BamA Mouse polyclonal antibodies were obtained from Dr. N. Noinaj [14]
α-FbpA Rabbit polyclonal antibodies were obtained from from Dr. A. B. Schryvers [15]
α-PgaB Rabbit polyclonal antibodies were obtained from Dr. D. Little and Dr. L. Howell This study α-LbpB Rabbit polyclonal antibodies were obtained from Dr. M. Joanne Lemieux [16]
α-6x His Mouse anti-6x His monoclonal antibody Pierce, MA121315 α-GST Rabbit anti-GST polyclonal antibody Cell Signalling, #2622
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DOI: 10.1038/NMICROBIOL.2016.9 SUPPLEMENTARY INFORMATION