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