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Lock and Key Authorized Replication System

Lock and Key Authorized Replication System. Research Talk 3 / 11 /1 4 Long Chen. Today’s Biotechnology. More than 200 new therapies and vaccines . 400 drug products and vaccines in clinical trials.

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Lock and Key Authorized Replication System

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  1. Lock and Key Authorized Replication System • Research Talk • 3/11/14 • Long Chen

  2. Today’s Biotechnology • More than 200 new therapies and vaccines. • 400 drug products and vaccines in clinical trials. • Biotechnology innovations are increasing food supplies, reducing damage to the environment, conserving natural resources of land, water and nutrients, and increasing farm income in economies worldwide. • Biotech innovation is cleaning our environment and fighting global climate change by reducing our dependence on petroleum and fossil fuels.

  3. Biotech Companies: IP dependent • Heavily dependent on scientific discoveries, bio-technologies and innovative tools. • Cost of innovation is high, protection of intellectual property is required for profitability. • Failure rate is high. A successful drug : ~$1-5 billion + 10-15 years

  4. Bio-espionage • In 1997, two U.S. citizens were arrested by the FBI and charged with attempting to steal the process for culturing Taxol from plant cells. • In 2002, a pair of medical researchers were arrested in San Diego for allegedly stealing genetic material and laboratory equipment from Harvard Medical School. • In December 2013, a corporate agriculture espionage case happened in Iowa. A Chinese man is accused of stealing highly valuable inbred corn seeds.

  5. Deficiencies of DNA Protection

  6. “GeneGuard” Prevents DNA replication in an unauthorized host. Enables DNA replication in an authorized host. No addition of chemical inducer needed. Many specific, orthogonal Lock and Key variants Protect high-value plasmid from being illegally reproduced and unexpected spread.

  7. How does Lock and Key system work? Wild Type Cis-acting “R-Loop”“G-Cluster” Synthetic Trans-acting

  8. Stages of L&K development • Proof of principle • Engineer multiple Lock and Key variants • Directed-evolution of L&K variants • Create Authorized Host • Application of Lock and Key System

  9. Experimental set-up In E.colipir116, the R6K origin is supposed to have a copy number up to 250. http://www.frankwu.com/R6K.html The R6K origin does not function in E. coli DH10B, allowing us to characterize synthetic origin.

  10. Proof of Principle • Removed the RNAII, leaving a truncated ColE1 origin only. The RNA II primer is essential for plasmid replication. E. coli pir116 E. coli DH10B

  11. Proof of Principle • Removed truncated ColE1 origin, leaving a relocated wt-RNAII only.

  12. Proof of Principle • We inserted 4 to 42 polyadenylation at 3’ end of wt-RNAII to deplete its self-sufficiency. http://mcb.asm.org/content/29/11/3124.full

  13. Proof of Principle • When Lock#X and Key#X are both present, the plasmid get replicated.

  14. Design Synthetic RNAII and Origin of Replication Minimize cis-acting replication (truncation and poly A insertion) Maximize trans-acting replication http://www.sciencedirect.com/science/article/pii/0092867486904915

  15. Module of Designing LOCK and KEY Variants Minimize cis-acting replication (truncation and Poly A insertion) Maximize trans-acting replication Synthetic RNA II and Synthetic Origin of Replication

  16. Design 37 Variants

  17. Clone and Evaluate 37 Lock and Key Variants LOCK#X + KEY#X version LOCK# Only version

  18. Study on TOP Four Lock and Key Variants Construct a dual-plasmid testing system The rest part?

  19. Directed-evolution on Lock and Key Complete process of Lock and Key directed-evolution

  20. Outputs of directed-evolution on Lock and Key

  21. Upgrading RNAII After identifying all the mutations from third round of directed-evolution, we get a new version of RNA II primer with known beneficial point mutations. Then we cloned back the other three Top Four rescue sequence and got a series of upgraded Top Four variants. The poly A section has dropped to 34 As. No mutations in rescue sequence. Upgraded RNA II primer

  22. Efficacy of directed-evolution on Lock and Key Before Directed-evolution After Directed-evolution

  23. Creating Authorized Hosts: Three Versions Integration of upgraded RNA II primers to the genome of E. coli. KanR H2 H1 RNA II primer KanR H1 RNA II primer H2 H1 H2 tonB yciL tonB KanR RNA II primer yciL

  24. Characterization and Comparison of Authorized Strains

  25. Application of Lock and Key System http://www.sciencedirect.com/science/article/pii/S1074552103001030

  26. Quantitative Analysis RNA―cDNA Rt-qPCR ? ? ? ? Total DNA Rt-qPCR

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