Culturing Escherichia coli cells

Scope of the method

Alternative method relates to
  • Other
Recombinant DNA technology
Alternative method is situated in
  • Basic Research
  • Regulatory use - Routine production
Type of alternative method
  • In vitro - Ex vivo
This method makes use of
  • Other (e.g. bacteria)
Escherichia coli, DH5alpha, C43 (DE3), BL21 (DE3)

Description

Method keywords
  • bacterial cells
  • cell culture
  • protein expression
  • E. coli
  • prokaryote
Scientific area keywords
  • microbiology
  • biotechnology
  • Recombinant DNA technology
Method description

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.

Lab equipment
  • Biosafety cabinet
  • Bunsen burner
  • Petri dishes
Method status
  • Still in development
  • History of use
  • Internally validated

Pros, cons & Future potential

Advantages
  • Fast growth kinetics (doubling time 20 mins)
  • High cell density cultures are easily achieved
  • Readily available and inexpensive components for media
  • Easy transformation
Challenges

No post-translational modifications (i.e. prokaryote)

Future & Other applications

Every researcher that will need a prufied protein can obtain it in a recombinant form.

References, associated documents and other information

Associated documents

Contact person

Jessie Neuckermans

Organisations

Vrije Universiteit Brussel
Pharmaceutical and Pharmacological Sciences (FARM)
In Vitro Toxicology and Dermato-cosmetology
Belgium