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Sticky Bacteria… Bacteri-adhesive… Cling- E. coli…

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

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Sticky Bacteria… Bacteri-adhesive… Cling- E. coli…

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

  2. 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

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

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

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

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

  7. 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)

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

  9. 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

  10. Ligations: 2 hr vs. overnight?

  11. LppOmpA1 LppOmpA2 His6 Strep2 His6 Strep2 - + - + - + - + Protein gel of LppOmpA1-His6 and LppOmpA-Strep2 188kD 62 49 38 28 18 14 6 3

  12. 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

  13. iGEM Mafia • Kevin was an innocent man wrongly accused and executed • Alex is God, and therefore cannot be killed • 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.

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