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Advanced Techniques for Fusion of Synthetic Oligos into Loop Insertions & Protein Termini

Learn fusion methods for synthetic oligos with OmpA & AIDA1, including loop insertion and C/N terminus fusion. Explore primer design, PCR digestion, ligation, and transform motif for gene insertion points.

jamal-richard
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Advanced Techniques for Fusion of Synthetic Oligos into Loop Insertions & Protein Termini

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Presentation Transcript

  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 Beads

  4. AIDA results

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

  6. E X S P P E M Loop Insertion: PCR products Pre-loop1 OmpA OmpA Portion with Modified loop1 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. E X S P N Gene Design

  10. X P N P Gene Design: Operations M M

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

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

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