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. Design and validation of MDS 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: Deletions

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!

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

13. ONLY IS MUTAGENESIS NOT POSSIBLE!

14. Induction of cycA mutations in MG1655 and MDS41

15. PLASMID STABILITY – pCTXVP60

16. PLASMID STABILITY – pT-ITR

17. PLASMID STABILITY

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

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., 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.htmlhttp://www.news.wisc.edu/newsphotos/perna.html  S5: http://www.scarabgenomics.com/pdfs/cleangenome.pdfhttp://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?

23. THANK YOU!

24. Referencec : www.slideshare.com