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

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 ES XP Pre-loop1 OmpA OmpA Portion with Modified loop1 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