Lidstrom:Autoinduction Media: Difference between revisions
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== References == | == References == | ||
* '''Original Paper:''' Studier FW (2005), Protein Production by Auto-Induction in High-Density Shaking Cultures. Protein Expr. Purif. 41(1): 207–234. <-- we make media ZYM-5052. | * '''Original Paper:''' Studier FW (2005), Protein Production by Auto-Induction in High-Density Shaking Cultures. Protein Expr. Purif. 41(1): 207–234. <-- we make media ZYM-5052. | ||
** A more practical recap of the original paper by the same author 12 years later: '''[http://link.springer.com/protocol/10.1007%2F978-1-62703-691-7_2#page-1 Stable Expression Clones and Auto-Induction for Protein Production in E. coli]''' ([https://www.bnl.gov/biosciences/staff/Studier/files/MMB%20Auto-Induction%20Chapter(13-09-04).pdf free version (?)]) | |||
* '''Practical summary slides:''' Imperial college .ppt slides: http://www3.imperial.ac.uk/pls/portallive/docs/1/15699698.PPT | * '''Practical summary slides:''' Imperial college .ppt slides: http://www3.imperial.ac.uk/pls/portallive/docs/1/15699698.PPT | ||
== Comparing autoinduction media to TB induced with IPTG == | == Comparing autoinduction media to TB induced with IPTG == | ||
[[User:Janet B. Matsen|Janet]] did an experiment in 12/2014 comparing cell pellets of [http://www.rcsb.org/pdb/explore/explore.do?structureId=3k9d His-tagged ADH] grown in autoinduction media to the same culture grown in TB and induced with IPTG as part of [https://docs.google.com/spreadsheets/d/1kT8DU30LBeUHHZW39FSRBd7QmYBeBa6IZ5u4ez5Jayo/edit?usp=sharing this experiment]. The autoinduced culture had comparable amounts of protein as seen by SDS-PAGE, and yielded more protein upon Co-NTA purification. | [[User:Janet B. Matsen|Janet]] did an experiment in 12/2014 comparing cell pellets of [http://www.rcsb.org/pdb/explore/explore.do?structureId=3k9d His-tagged ADH] grown in autoinduction media to the same culture grown in TB and induced with IPTG as part of [https://docs.google.com/spreadsheets/d/1kT8DU30LBeUHHZW39FSRBd7QmYBeBa6IZ5u4ez5Jayo/edit?usp=sharing this experiment]. The autoinduced culture had comparable amounts of protein as seen by SDS-PAGE, and yielded more protein upon Co-NTA purification. | ||
* [[image:141219_SDS-PAGE_showing_efficacy_of_autoinduced_ADH.png|upright=1.5|thumb|center|Autoinduction Media worked as well as IPTG.]] | * [[image:141219_SDS-PAGE_showing_efficacy_of_autoinduced_ADH.png|upright=1.5|thumb|center|Autoinduction Media worked as well as IPTG.]] | ||
Revision as of 12:23, 24 March 2015
Return: Protocols
Recipe:
Autoinduction Media recipe from Studier 2005. 
Autoinduction Media antibiotic specificiations. - Recipe in action -JM
Why Autoinduction Media?
Instead of inducing a T7 promoter with IPTG, autoinduction media can be used. Glucose, glycerol, and fructose are added to a buffered yeast broth to make autoinduction media. As the E. coli cultures grow, they consume the glucose first. As the glucose runs out, they are forced to use the fructose which drives expression of the T7 promoter. Glycerol helps support growth without inhibiting T7 protein expression.
The advantages are:
- you don't have to monitor OD of the cultures
- you can go straight from -80oC to protein purification in 1 day instead of two
- great for library screening where cultures have different growth rates
References
- Original Paper: Studier FW (2005), Protein Production by Auto-Induction in High-Density Shaking Cultures. Protein Expr. Purif. 41(1): 207–234. <-- we make media ZYM-5052.
- A more practical recap of the original paper by the same author 12 years later: Stable Expression Clones and Auto-Induction for Protein Production in E. coli (free version (?))
- Practical summary slides: Imperial college .ppt slides: http://www3.imperial.ac.uk/pls/portallive/docs/1/15699698.PPT
Comparing autoinduction media to TB induced with IPTG
Janet did an experiment in 12/2014 comparing cell pellets of His-tagged ADH grown in autoinduction media to the same culture grown in TB and induced with IPTG as part of this experiment. The autoinduced culture had comparable amounts of protein as seen by SDS-PAGE, and yielded more protein upon Co-NTA purification.
Autoinduction Media worked as well as IPTG.
