REDUCED GENOME E.coli
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Transcript REDUCED GENOME E.coli
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
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
Design and validation of MDS
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!
Adaptation of MDS41 and MG1655 to Salicin/Minimal Medium
● : MG1655
▼:
MDS41
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
A. MDS41 in minimal growth medium
■ : optical density (left scale)
● : DCW (left scale)
▼:
glucose concentration (right scale)
B. CAT expression in MDS41 and MG1655
■ : MG1655
● and▼: MDS41 duplicates
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 reducedgenome 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):13341.
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?
Referencec : www.slideshare.com