Unless otherwise indicated, all chemicals and reagents were purchased from Sigma-Aldrich. Restriction enzymes, KOD DNA polymerase, Q5 2X Master Mix, Phusion 2X Master Mix, and T4 polynucleotide kinase were purchased from New England Biolabs. The In-fusion HD Cloning Plus kit was purchased from Takara Bio USA. Oligonucleotide primers were purchased from Integrated DNA Technologies (IDT). Culture media were purchased from Becton Dickinson. Streptococcus pneumoniae ΔmurMN Lipid II was isolated from cells as described previously (53 (link), 54 (link)). Lipid II was labeled with ATTO488 as previously described (11 (link)). S. aureus SgtBY181D was expressed and purified as previously reported (55 (link)). Genomic DNA was isolated using a Wizard Genomic DNA Purification kit (Promega).
Protocol detail
Fluorescent Lipid II Assay for Pneumococcal Cell Wall Synthesis
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Q5 2X Master Mix
Phusion 2X Master Mix
T4 polynucleotide kinase
In-fusion HD Cloning Plus kit
Oligonucleotide primers
Wizard Genomic DNA Purification kit
Cells
Culture media
Dna polymerase
Genomic
Lipid ii
Oligonucleotide primers
Polynucleotide kinase
Promega
Restriction enzymes
S aureus
Streptococcus pneumoniae
Corresponding Organization :
Other organizations : Boston VA Research Institute, Harvard University
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Variable analysis
independent variables
- Lipid II isolation from Streptococcus pneumoniae Δmurniurty cells
- Labeling of Lipid II with ATTO488
- Expression and purification of Staphylococcus aureus SgtB^Y181D
- Not explicitly mentioned
- Chemicals and reagents purchased from Sigma-Aldrich, New England Biolabs, Takara Bio USA, and Integrated DNA Technologies (IDT)
- Culture media purchased from Becton Dickinson
- Genomic DNA isolation using Wizard Genomic DNA Purification kit (Promega)
- Positive control: Not specified
- Negative control: Not specified
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