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CLEAN GENOME E. coli – multiple deletion strains

CLEAN GENOME E. coli – multiple deletion strains. Gulpreet Kaur Microbial Biotechnology, Fall 2011. A bit of history…. Fredrick Blattner : 1997 - published complete genome of E.coli-K12 strain 2002 - engineered reduced E. coli genome -developed Scarab Genomics

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CLEAN GENOME E. coli – multiple deletion strains

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  1. CLEAN GENOME E. coli – multiple deletion strains 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 genome E. coli

  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: 20%

  6. What to delete? • Genes specific for some environments • Potential pathogenicity genes • DNA sequence repeats • Mobile DNA elements that mediate recombination events • Insertion Sequences • 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. flexneri2457T 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: Deletions Design and validation of MDS

  8. Comparison among strains

  9. TRANSFORMATION EFFICIENCIES • Efficiencies of MDS42 were twice that of MG1655 • Efficiencies of MDS42 were comparable to DH10B

  10. NO IS SEQUENCES!

  11. NO IS SEQUENCES!

  12. NO IS-MEDIATED MUTAGENESIS! Adaptation of MDS41 and MG1655 to Salicin/Minimal Medium ● : MG1655 ▼: MDS41

  13. ONLY IS MUTAGENESIS NOT POSSIBLE!

  14. ONLY IS MUTAGENESIS NOT POSSIBLE! Induction of cycA mutations in MG1655 and MDS41

  15. PLASMID STABILITY – pCTXVP60

  16. PLASMID STABILITY – pT-ITR

  17. PLASMID STABILITY

  18. GROWTH RATES A. MDS41 in minimal growth medium B. CAT expression in MDS41 and MG1655 ■ : optical density (left scale) ● : DCW(left scale) ▼: glucose concentration (right scale) ■ : MG1655 ● and▼: MDS41 duplicates

  19. CONCLUSIONS The 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., BlattnerF.R., Posfai G. 2002. Engineering a reduced Escherichia coli genome. Genome Res. 12(4):640-7. • BlattnerF.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, BlattnerFR, 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, BlattnerFR, 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, BlattnerFR, 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.   • UmenhofferK, Fehér T, Balikó G, Ayaydin F, Pósfai J, BlattnerFR, 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?

  23. THANK YOU!

  24. Referencec : www.slideshare.com

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