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

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:

6. What to delete?
 Genes specific for some environments
 Potential pathogenicity genes
 DNA sequence repeats
 Mobile DNA elements that mediate
recombination events
 InsertionSequences
 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. 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

8. Comparison among strains

9. TRANSFORMATION
EFFICIENCIES
 Efficiencies of MDS42 were twice that of
MG1655

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 B. CAT expression in MDS41 and
medium MG1655
: optical density (left scale)
: MG1655
: DCW (left scale)
and : MDS41 duplicates
: glucose concentration (right

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.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, 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?