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

• 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

QuickTime™ and aTIFF (LZW) decompressorare needed to see this picture.QuickTime™ and aTIFF (LZW) decompressorare needed to see this picture.

Without amoxycilin (1KE4) and with amoxycillin (1LL9)

Part of MSA

QuickTime™ and aTIFF (LZW) decompressorare needed to see this picture.

MSA on Structure

QuickTime™ and aTIFF (LZW) decompressorare needed to see this picture.