IGEM 2007 Cell Surface Re-engineering SKS. Outline Cell surface re-engineering Preliminary Results Back to the Drawing Board Outlook.

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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 …and establishing quorum

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

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

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

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

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

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

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)

Biobrick Parts ES XP Pre-loop1 OmpA OmpA Portion with Modified loop1 E P E|P digested vector

2 step PCR results

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

Colony PCR 1 Kb lane

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