Sticky Bacteria… Bacteri-adhesive… Cling-E. coli… A working title Perry Tsai June 25, 2007.

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Presentation transcript:

Sticky Bacteria… Bacteri-adhesive… Cling-E. coli… A working title Perry Tsai June 25, 2007

The goals Develop a system for random peptide display on the bacterial surface, in order to screen for peptides that bind to a target with high affinity Use this system to identify consensus peptide sequences that bind to targets of interest

Lpp-OmpA membrane display periplasm extracellular outer membrane J. Francisco C. Earhart G. Georgiou 1992 Lpp (lipoprotein) signal peptide OmpA (outer membrane protein A) transmembrane domains Surface peptide

The construct BioBricks protein fusion Digest/ ligation LppOmpA1 or 2 in pET29b+ vector NheI PstI + Random 30nt oligo sequences SpeI PstI

Random peptide display and screening + Transformation Expression MACS (target with magnetic bead) FACS (target with fluorescent tag)

Sequence identification and targeting generalization … ctg gct ggt ttc … L A G F = Bacterial targeting Affinity purification Eukaryotic cell targeting Protein targeting Fluorescent tagging

Using known peptide interactions as positive controls His6 tag + nickel beads Strep2 tag + streptavidin beads PDZ-binding tag + GST PDZ domains PDZ domains + PDZ-binding tag (GFP)

Digest and gel isolation LppOmpA1 NheI/PstI LppOmpA2 NheI/PstI LppOmpA1 or 2 in pET29b+ vector NheI PstI

Amplifying the random library: PCR vs extension? SpeI PstI 5’ gcggctg ggag actagt nnb nnb nnb nnb nnb nnb nnb nnb nnb nnb gctagc tgataa ctgcag cccg 3’ PCR Extension

Ligations: 2 hr vs. overnight?

Protein gel of LppOmpA1-His6 and LppOmpA-Strep2 188kD LppOmpA1LppOmpA2 His6Strep2His6Strep

For the next week… Figure out ways to increase ligation and transformation efficiency Continue brainstorming  Targets  Signal transduction systems Testing MACS magnetic bead assay using positive controls

iGEM Mafia Bill, George, and Nick have been killed by the Mafia Perry and Alain were sniffed out as members of the Mafia and appropriately lynched; one still remains. Kevin was an innocent man wrongly accused and executed Alex is God, and therefore cannot be killed