clean genome e. coli – multiple deletion strains gulpreet kaur microbial biotechnology, fall 2011
TRANSCRIPT
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
2006 - emergent properties of reduced genome E. coli
Why E.Coli K-12?
Vast knowledge on its genomic organization
Commonly used for research and metabolite production
Popular strains – MG1655 and W3110
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
What to delete?
Backbone genome: 3.71Mb
Total genome targeted to be deleted: 20%
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
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 genomes1: RS2182: CFT0733: S. flexneri 2457T4: O157:H7 EDL9335: DH10B Ring 6: Deletion targetsRed: MDS12Yellow: MDS41Green: MDS 42Purple: MDS43Ring 7: Native IS elementsRing 8: Confirmation of deletion in MDS43Red: Genome presentGreen: Deletions
Comparison among strains
TRANSFORMATION EFFICIENCIES
Efficiencies of MDS42 were twice that of MG1655 Efficiencies of MDS42 were comparable to DH10B
NO IS SEQUENCES!
NO IS SEQUENCES!
NO IS-MEDIATED MUTAGENESIS!
● :
MG1655
▼: MDS41
Adaptation of MDS41 and MG1655 to Salicin/Minimal
Medium
ONLY IS MUTAGENESIS NOT POSSIBLE!
ONLY IS MUTAGENESIS NOT POSSIBLE!
Induction of cycA mutations in MG1655 and MDS41
PLASMID STABILITY – pCTXVP60
PLASMID STABILITY – pT-ITR
PLASMID STABILITY
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
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
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)
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.
QUESTIONS?
THANK YOU!
Referencec : www.slideshare.com