Techniques Fusion of ds synthetic oligos to the C terminus of OmpA Fusion of ds synthetic oligos to the N terminus of AIDA1 Fusion of tags & randomers to extracellular portion of OmpA loop 1 (loop insertion) Mistake here with AIDA1 – we did not make additions to the C terminus

Fusion of tags to N-terminus of AIDA-1

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

Loop Insertion PCR product digestion & ligation Gene design Primer design Digest-Ligate-Transform motif Gene design Created insertion points for compatible synthetic constructs