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iGEM 2007

iGEM 2007. Cell Surface Re-engineering SKS. Outline. Cell surface re-engineering Preliminary Results Back to the Drawing Board Outlook. Cell surface Re-engineering. Expressing random peptide sequences on the E. coli cell surface creates new opportunities for: Ligand identification

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iGEM 2007

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  1. iGEM 2007 Cell Surface Re-engineering SKS

  2. Outline • Cell surface re-engineering • Preliminary Results • Back to the Drawing Board • Outlook

  3. Cell surface Re-engineering • Expressing random peptide sequences on the E. coli cell surface creates new opportunities for: • Ligand identification • …and establishing quorum

  4. Outline • Cell Surface Re-engineering • Preliminary Results • Back to the Drawing Board • Outlook

  5. Procedure • Ligated CDS for his and strep downstream of the OmpA gene • Ligated construct into a vector & transformed resultant plasmid & induced protein expression with IPTG • Ran a protein gel

  6. Before induction Before induction His Strep Strep His His Strep His Strep ladder After induction Protein Gel Results *Blue text boxes represent the tags fused to OmpA

  7. Cell Sorting • Realized low yields after running a MACS column and FACS considered an alternative strategy

  8. Outline • Cell surface re-engineering • Preliminary Results • Back to the Drawing Board • Outlook

  9. Alternative Strategy • Affix the RNS to the side of loop 1 exposed to extracellular space • Two-step PCR • Use an NheI ‘surgical’ site

  10. Two-step PCR • Create two products: • Product 1 has coding sequence for the beginning of OmpA until just before extracellular loop 1 (about 150bps) • Product 2 has a substitution replacing part of the extracellular portion of loop 1 and going on till the end of OmpA (about 950 bps)

  11. Biobrick Parts Pre-loop1 OmpA E S OmpA Portion with Modified loop1 X P E P E|P digested vector

  12. 2 step PCR results

  13. Ligations & Transformations • Realized transformant colonies of: • OmpA with strep • OmpA with His • OmpA with a 10-mer • OmpA with a 15-mer

  14. Colony PCR 1 Kb lane

  15. Outlook • Sequencing • Transform into BL23 DE3 cells • MACS & FACS for the cells with his & strep • Find consensus sequences for targets

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