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!