Culturing Escherichia coli cells
Scope of the method
- Other
- Basic Research
- Regulatory use - Routine production
- In vitro - Ex vivo
- Other (e.g. bacteria)
Description
- bacterial cells
- cell culture
- protein expression
- E. coli
- prokaryote
- microbiology
- biotechnology
- Recombinant DNA technology
E. coli is one of the organisms of choice for the production of recombinant proteins. DH5 alpha cells are commonly used for maintenance, propagation and mutation, whilst BL21(DE3) and C43(DE3) are mainly used for expression of the transgene. The advantage of C43(DE3) is that is used to produce proteins that are expressed poorly in BL21 (DE3) or that are very toxic to the host organism. All strains can be cultured in Lysogeny Broth (LB) medium or LB agar plates with an appropiate antibiotic for positive selection of the clones. For induction of protein expression, isopropyl-b-thiogalactoside (IPTG) in a concentration of 0.2 mM - 1 mM can be used. In case you have a problems with leaky expression, 1 % w/v glucose can be added to the LB medium for excellent growth of the bacteria. Transformation of the cells can be achieved by heat shock or electroporation.
- Biosafety cabinet ;
- Bunsen burner ;
- Petri dishes.
- Still in development
- History of use
- Internally validated
Pros, cons & Future potential
- Fast growth kinetics (doubling time 20 mins) ;
- High cell density cultures are easily achieved ;
- Readily available and inexpensive components for media ;
- Easy transformation.
No post-translational modifications (i.e. prokaryote).
Every researcher that will need a prufied protein can obtain it in a recombinant form.
