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Clean genome ecoli

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  • 1. CLEAN GENOME E. COLI –MULTIPLE DELETIONSTRAINS Gulpreet Kaur Microbial Biotechnology, Fall 2011
  • 2. A bit of history…Fredrick Blattner: 1997 - published complete genome of E.coli-K12 strain 2002 - engineered reduced E. coli genome - developed Scarab Genomics 2006 - emergent properties of reduced
  • 3. Why E.Coli K-12? Vast knowledge on its genomic organization Commonly used for research and metabolite production Popular strains – MG1655 and W3110
  • 4. Why reduce the genome? Problems in using E. coli K-12 strains:  Loss of desired gene over time  Mutation of desired gene  Low protein productivity  Lack of purity in product  Batch-to-batch variations  High production costs
  • 5. What to delete?  Backbone genome: 3.71Mb  Total genome targeted to be deleted:
  • 6. What to delete? Genes specific for some environments Potential pathogenicity genes DNA sequence repeats Mobile DNA elements that mediate recombination events  InsertionSequences  Transposases, Integrases  Defective phage remnants
  • 7. Outer Ring: E. coli K-12 Inner rings: (from center to outwards) 1-5: regions of E. coli K- 12 absent in other genomes 1: RS218 2: CFT073 3: S. flexneri 2457T 4: O157:H7 EDL933 5: DH10B Ring 6: Deletion targets Red: MDS12 Yellow: MDS41 Green: MDS 42 Purple: MDS43 Ring 7: Native IS elements Ring 8: Confirmation of deletion in MDS43 Red: Genome present Green: DeletionsDesign and validation of MDS
  • 8. Comparison among strains
  • 9. TRANSFORMATION EFFICIENCIES Efficiencies of MDS42 were twice that of MG1655
  • 10. NO IS SEQUENCES!
  • 11. NO IS SEQUENCES!
  • 12. NO IS-MEDIATEDMUTAGENESIS! Adaptation of MDS41 and MG1655 to Salicin/Minimal Medium : MG1655 : MDS41
  • 13. ONLY IS MUTAGENESIS NOTPOSSIBLE!
  • 14. ONLY IS MUTAGENESIS NOTPOSSIBLE! Induction of cycA mutations in MG1655 and MDS41
  • 15. PLASMID STABILITY –pCTXVP60
  • 16. PLASMID STABILITY – pT-ITR
  • 17. PLASMID STABILITY
  • 18. GROWTH RATESA. MDS41 in minimal growth B. CAT expression in MDS41 andmedium MG1655 : optical density (left scale) : MG1655 : DCW (left scale) and : MDS41 duplicates : glucose concentration (right
  • 19. CONCLUSIONSThe strains have the following: Enhanced transformation efficiency Reduced mutability Increased plasmid stability Normal growth rates Can me used as ‘chassis’ for metabolite production
  • 20. BIBLIOGRAPHY Posfai G. et. al., 2006. Emergent properties of reduced-genome Escherichia coli. Science 312, 1044-1046. Kolisnychenko V., Plunkett G. III, Herring C.D., Feher T. Posfai J., Blattner F.R., Posfai G. 2002. Engineering a reduced Escherichia coli genome. Genome Res. 12(4):640-7. Blattner F.R. et. al., 1997. The Complete Genome Sequence of Escherichia coli K-12. Science 277, 1453-1469.Pictures, Figures, Tables: S2: http://www.news.wisc.edu/newsphotos/perna.html S5: http://www.scarabgenomics.com/pdfs/cleangenome.pdf S7,8,9,12,14,18: Posfai G. et. al., 2006. Emergent properties of reduced-genome Escherichia coli. Science 312, 1044-1046 S11, 17: Posfai G. et. al., 2006. Emergent properties of reduced-genome Escherichia coli. Science 312, 1044-1046 (supporting online material)
  • 21. FURTHER READING… Sung BH, Lee CH, Yu BJ, Lee JH, Lee JY, Kim MS, Blattner FR, Kim SC. Development of a biofilm production-deficient Escherichia coli strain as a host for biotechnological applications. Appl Environ Microbiol. 2006 May;72(5):3336-42. Sharma SS, Blattner FR, Harcum SW. Recombinant protein production in an Escherichia coli reduced genome strain. Metab Eng. 2007 Mar;9(2):133- 41. Lee JH, Sung BH, Kim MS, Blattner FR, Yoon BH, Kim JH, Kim SC. Metabolic engineering of a reduced-genome strain of Escherichia coli for L-threonine production. Microb Cell Fact. 2009 Jan 7;8:2. Umenhoffer K, Fehér T, Balikó G, Ayaydin F, Pósfai J, Blattner FR, Pósfai G. Reduced evolvability of Escherichia coli MDS42, an IS-less cellular chassis for molecular and synthetic biology applications. Microb Cell Fact. 2010 May 21;9:38.
  • 22. QUESTIONS?