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

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. Outline of Laboratory

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

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).

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.

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

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

Part of MSA

MSA on Structure