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Engineering Competency in E. coli: A Synthetic Biology Approach

This study explores the synthetic biology approach to enhance competency in E. coli, focusing on the methods for inserting new DNA, specifically the insulin gene. It discusses natural and induced competency mechanisms, highlighting the role of the ComK protein in activating genes responsible for late competency. Through a detailed analysis of controlled expression systems, this research aims to build a testable framework for assessing the competency of E. coli strains and their ability to uptake plasmids effectively. Contributions from various research initiatives underscore the collaborative effort behind this work.

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Engineering Competency in E. coli: A Synthetic Biology Approach

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  1. A Synthetic Biology Approach To Engineered Competency in E. coli Samantha Simpson Genomics 4/8/09

  2. Why Would You Want To Insert New DNA into E. coli? Insulin Gene Extracted Plasmid

  3. My Focus Insulin Gene Extracted Plasmid

  4. A Closer Look at Taking Up New DNA

  5. What Is Competency?

  6. How does natural competency work?

  7. How does ComK activate late competency genes? ComK Late Competency Gene K-box promoter

  8. How are cells made competent? • Natural Competency in B. subtilis • Controlled by ComK • Induced Competency in E. coli Both 48 genes Microarray 57 genes K-boxes 1014

  9. Is natural competency feasible in E. coli?

  10. Is natural competency feasible in E. coli? K-Box to gene distance not consistent.

  11. Building a Testable System } 600 bps ComK ComK MWM High Copy AmpR 1018 bp 506 bp 396 bp 344 bp 298 bp

  12. Building a Testable System RBS ComK GFP pKBox Low Copy High Copy AmpR KanR

  13. Testing Fluorescence p < 0.02

  14. Building a Testable System RBS RBS ComK GFP pKBox Low Copy High Copy AmpR KanR

  15. RBS’s: Not Consistent

  16. Promoters: Consistent

  17. Building a Competent System ComK Low Copy KanR

  18. Testing Competency ComK / Kan Pellet Cells RFP / Amp LB + Kan/Amp

  19. Conclusions In E. coli: In B. subtilis: pKBox + ComK

  20. Acknowledgments • Davidson Research Initiative • National Science Foundation • Howard Hughes Medical Institute • James G. Martin Genomics Program • Dr. Heyer, Dr. Campbell and Dr. Denham • Fellow lab workers • Friends and Family

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