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)

Beads

Pre-assay plates

AIDA results

Loop Insertion

• PCR product digestion & ligation – Primer design– Digest-Ligate-Transform motif

• Gene design– Insertion points created for inserting synthetic

constructs

Loop Insertion: PCR products

E S

Pre-loop1 OmpA

OmpA Portion with Modified loop1

E P

E|P digested vector

X P

Complete Plasmid

M

PCR productsLane1: 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 PX SN

Gene Design: Operations

N P

X PM

M

Gene Design: Operations

N NX S

M

M

M

M

His/strep tags OR randomers

PCR Results

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