Delphi Genetics SA How to Render Antibiotics Obsolete Using ...

Delphi Genetics SA 

How to Render Antibiotics Obsolete Using 

Bacterial Selection Modules for Efficient 

Protein Production in E. coli 

Philippe Gabant (PhD) 

Delphi Genetics SA, Gosselies (Be) 

Biofit: October 26 -27, 2010 (Lille) 

© Delphi Genetics S.A. 

www.delphigenetics.com 

1

Company Overview 

• Spin Off of the Université Libre de Bruxelles 

(ULB) founded in 2001 by the inventors of the 

technologies 

• The experts in Poison-Antidote technologies 

• Broad international patent portfolio for key 

technologies 

• Market: Genetic engineering and 

Biopharmaceuticals 

• 6 Products on the Market (Kits), Expert Service 

Provider 

• 14 employees (5 PhDs) 



2

Company Activities 

•Expert service provider for gene expression, DNA cloning and 

recombinant protein production. 

•Development of research kits for Molecular Cloning and Protein 

Expression based on proprietary technologies. Products are sold 

directly by Delphi Genetics and distributed globally via a distribution 

network 


•Licensing of technology for biopharmaceutical applications 


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E. Coli is still the workhorse for Molecular Biologists 

Excellent Tool for intracellular production: proteins and pDNA 

Low costs of goods, robust platform, high titers achievable 

BUT: Antibiotics markers are used during cloning (clone 

selection) and production (plasmid stabilization) 



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Industrial drawbacks of the antibiotic markers in bioproduction: 

• Antibiotic resistant genes (markers) represent a burden of 

energy for the bacterial host leading to a decrease of 

productivity, therefore not always compatible for production of 

difficult protein; 

• Regulatory issues, e.g. for vaccinal (i.e. injectable) pDNA or 

therapeutic recombinant proteins: only few markers tolerated. 

Total absence of antibiotics need to be achieved in the final 

product. 

• Environmental issues: use of antibiotics in large fermentors, 

generation of bacteria harboring resistance genes. 

An alternative to antibiotic markers is needed to i) increase the 

production yield and ii) make bioproductions more in line with 

Regulatory Authorities suggestions 



5

Delphi Genetics’ Solution (1) 

Delphi Genetic’s proprietary Staby TM technology adresses these 

issues: 

New selection and stabilization tool 

o Based on naturally occurring poison-antidote genes 

O/P 

ccdA 

Antidote 

ccdB 

Poison 

o The poison gene ccdB targets an essential bacterial 

process: DNA Replication (DNAgyrase) 

o The antidote ccdA interacts with ccdB and blocks its 

lethal activity by transcriptional silencing and via 

protein-protein interactions 


Powerful alternative to antibiotic markers for clone 

selection and plasmid stabilization 


6

Delphi Genetics’ Solution (2) 

Principle of the technology : 

– Poison gene integrated in the bacterial chromosome 

– Promoter of the poison is repressed by the antidote 

– Antidote gene on the plasmid 

– Bacteria losing the plasmid will die as the action of 

the poison is not inhibited 

– Creates addiction to the plasmid (w/o antibiotics) 

Applications : 

Bioproduction of: 

• Protein 

• Plasmid DNA 

© Delphi Genetics S.A.

Applications of the technology : Plasmid stabilization (1) 

Overview 

Plasmid 

Antidote (CcdA) 

Poison (CcdB) 

Target 

Gene of interest 

Bacterial growth, 

overexpression of the gene 

of interest 

Plasmid 



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Applications of the technology : Plasmid stabilisation (2) 

Plasmid Map 

•pET based vector 

backbone to which the 

ccdA gene has been 

added. 

•Compatible with current 

expression systems 

including T7 



9


Protein production 

Protein production with StabyExpress TM : 

1 2 3 4 5 6 7 8 9 

1 : SE1 TraGhis not induced (2µl) 

2 : BL21DE3 TraGhis not induced (2µl) 

3 : SE1 TraGhis induced (2h) (2µl) 

4 : BL21DE3 TraGhis induced (2h) (2µl) 

5 : Size 

6 : idem 1 (5µl) 

7 : idem 2 (5µl) 

8 : idem 3 (5µl) 

9 : idem 4 (5µl) 


After 30 generations, the protein production 

level is 3 to 5 times higher using the 

StabyExpress TM system than in the case of 

BL21DE3 (after only 2hours of induction of a 

non-toxic protein). 


10



11


DNA production 

DNA production with Staby TM DNA: 

1 2 3 4 5 

1 : Plasmid-DNA production using 

typical E. coli strain. Digestion by 

BamHI 

2 : Plasmid-DNA extraction using 

Staby TM DNA. Digestion by BamHI 

3 and 4: DNAs digested by StyI 

5 : Size 

After 30 generations, the DNA-extraction yield is 5 

to 10 times higher using StabyDNA TM than when 

using a typical E. Coli strain. 



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Benefits of the Staby TM technology 

• Higher yields of expressed proteins or DNA due to perfect plasmid 

stabilization and limitation of the burden of energy 

• No need to use antibiotics in the production process, thus meeting 

FDA and EMEA regulations and guidelines. The system Staby 

express system work with animal free medium 

• Scalable fermentation process, the stabilization technology is based 

solely on genetics elements and is thus readily scalable. The process 

is easily transferred from R&D to industrial scale fermentation thus 

creating an integrated platform for the production of Biomolecules in 

E. Coli 

• Any vector or E.Coli strain can be enhanced with the Staby TM 

technology 


An integrated platform for BioProcessing from R&D to industrial scale 

fermentation without the use of antibiotics or antibiotic-resistance genes 

Staby Express is licensed to: 


Thank you for your attention 

Delphi Genetics SA 

Tel : +32 71 25 10 00 

Fax: +32 71 37 60 57 

pgabant@delphigenetics.com 

Rue C. Ader 16 

B-6041 Gosselies, Belgium 



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Delphi Genetics SA How to Render Antibiotics Obsolete Using ...

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