Slide Collection SKS

Techniques Fusion of ds-synthetic oligos to the C terminus OmpA & AIDA1 Fusion to the N terminus AIDA1 Fusion to extracellular portion of loop 1 (loop insertion) OmpA

Fusion at C terminus: Bead Assay (His tag) Pre-assay plates C terminus addition of his to the ompA gene The top shows the plates prior to separation by cobalt beads Bottom is the beads – whish have the bact containing the ompAhis + sender (with RFP) The ratios (1:100) are the dilutions I made prior to the assay – so there is in fact enrichment. Beads

AIDA results

Loop Insertion PCR product digestion & ligation Gene design Primer design Digest-Ligate-Transform motif Gene design Insertion points created for inserting synthetic constructs

Loop Insertion: PCR products Pre-loop1 OmpA X P OmpA Portion with Modified loop1 E P M Complete Plasmid E|P digested vector

PCR products Lane1: Ladder Lane2: 1st portion OmpA Lane 3: strep2-OmpA portion 2 Lane 4: 6xHis-OmpA portion 2

PCR: final plasmid as a template Red line indicates the 1 kb band

Gene Design E P X S N

Gene Design: Operations M X P M

Gene Design: Operations His/strep tags OR randomers X S N M M M M

PCR Results Red line indicates the 1 kb line (7/8)