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Database to Benchtop: A study of ß-lactamase

Database to Benchtop: A study of ß-lactamase. Doug Welsh, Susan DeSimone, Mark Lubkowitz and Jill Salvo Based on: Stahelin et al., BAMBED, 31; 106-112, 2003. Outline of Laboratory. Use ß-lactamase gene Mutagenize active site Screen for effect on activity, or zone of inhibition

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Database to Benchtop: A study of ß-lactamase

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  1. Database to Benchtop:A study of ß-lactamase Doug Welsh, Susan DeSimone, Mark Lubkowitz and Jill Salvo Based on: Stahelin et al., BAMBED, 31; 106-112, 2003

  2. Outline of Laboratory • Use ß-lactamase gene • Mutagenize active site • Screen for effect on activity, or zone of inhibition • Generate crude lysate for colorometric assay. • Possibility to “hand-off” mutants to second class for purification and kinetics.

  3. BioInformatics Component • Using a pre-generated data set of beta-lactams, students select 5 and compare with known structure (Multiple Sequence Alignment) • Look for conserved regions, identify active site, domains?, motifs? • Possibly continue using MEME to motif search or Blast to look for related proteins (not ß-lactams), and go from there

  4. Wet Lab Details • Use mutagenic primer (a random primer would be really cool, but may give too many null activity mutants) over the active site that incorporates a restriction site change into the primer. • Students do PCR mutagenesis, rapid ligate, transform, pick colonies, prep DNA, and screen by restriction digest. • Crude lysate activity or zone of inhibition (allows screening of many possible mutants).

  5. Additional BI Component • Use Lasergene or other similar program to take sequence of plasmid, create mutagenic change, and generate virtual restriction digest pattern and gel.

  6. Selected Sequence(s) • E. coli ß lactamse chain B • Pseudomonas ß lactamase • Pyrococcus abyssi ß lactamase • E. coli ß lactamase chain a • Nostoc ß lactamase

  7. E. Coli ß-lactamase Without amoxycilin (1KE4) and with amoxycillin (1LL9)

  8. Part of MSA

  9. MSA on Structure

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