1. Slide Collection
SKS

2. 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

3. 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

4. AIDA results

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

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

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

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

9. Gene Design E P X S N

10. Gene Design: OperationsM X P M

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

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