Here’s a tip that you may find useful if you are expressing proteins in E.coli using a lac promoter-based expression system, e.g. pET, in LB medium (L-broth).
Lac expression systems are typically induced in the lab using IPTG (isopropyl-beta-D-thiogalacto- pyranoside), which is a non- hydrolysable analogue of lactose, the natural inducer of the lac operon.
Tight control of expression from the lac promoter, which is required if the protein being expressed is toxic to the E.coli host or for a variety of other reasons, is not possible when using LB because it contains lactose.
But how does lactose get into LB? Well, as you know, the main components of LB are tryptone and yeast extract.
Tryptone is basically a soup of peptides and amino acids that the cell can use directly for building proteins, or as a source of metabolisable carbon and nitrogen. It is produced by digesting casein, the most abundant protein in cow’s milk, with the proteolytic enzyme, trypsin.
So, tryptone is basically chopped up milk protein, and which sugar is a major component of milk? You’ve guessed it, lactose.
So that’s how lactose gets into LB, but thankfully there’s no need to throw the LB out of the window just yet because tight control of lac-based expression is still possible due to the wonders of catabolite repression.
Just add 0.3% glucose to the LB and expression from the lac-based promoter will be shut off like magic, giving you complete control once again.
Photo: sohvimus
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Nick Oswald
Nick is a molecular biologist-turned-publisher. After a PhD in Developmental Biology and an eclectic seven years in biotech he is now Editorial Manager of Neuroendocrinology and the founder and Editor-In-Chief of Bitesize Bio. You are welcome to connect with Nick on LinkedIn
wow..that’s a nifty trick! eventhough i studied about catabolite repression – i never thought to apply it this way. Cool tip Nick. Thanks.
You can also co-transform your cells with a pLysS plasmid, as this also ensures complete shutoff of the promoter, and helps lysing the cells when needed as well.
Liam – that’s a good tip too, but remember it’s only applicable to expression using the T7 promoter (not other lac-based promoters).
An article that underline this fact and give a method for protein expression, even toxic ones…
Just try it !
You can use this medium composition to reduce volumes into coli cultures for plasmid preparation or more…
Here the abstract :
Protein Expr Purif. 2005 May;41(1):207-34.Links
Protein production by auto-induction in high density shaking cultures.
Studier FW.
Biology Department, Brookhaven National Laboratory, Upton, NY 11973, USA. studier@bnl.gov
Inducible expression systems in which T7 RNA polymerase transcribes coding sequences cloned under control of a T7lac promoter efficiently produce a wide variety of proteins in Escherichia coli. Investigation of factors that affect stability, growth, and induction of T7 expression strains in shaking vessels led to the recognition that sporadic, unintended induction of expression in complex media, previously reported by others, is almost certainly caused by small amounts of lactose. Glucose prevents induction by lactose by well-studied mechanisms. Amino acids also inhibit induction by lactose during log-phase growth, and high rates of aeration inhibit induction at low lactose concentrations. These observations, and metabolic balancing of pH, allowed development of reliable non-inducing and auto-inducing media in which batch cultures grow to high densities. Expression strains grown to saturation in non-inducing media retain plasmid and remain fully viable for weeks in the refrigerator, making it easy to prepare many freezer stocks in parallel and use working stocks for an extended period. Auto-induction allows efficient screening of many clones in parallel for expression and solubility, as cultures have only to be inoculated and grown to saturation, and yields of target protein are typically several-fold higher than obtained by conventional IPTG induction. Auto-inducing media have been developed for labeling proteins with selenomethionine, 15N or 13C, and for production of target proteins by arabinose induction of T7 RNA polymerase from the pBAD promoter in BL21-AI. Selenomethionine labeling was equally efficient in the commonly used methionine auxotroph B834(DE3) (found to be metE) or the prototroph BL21(DE3).
PMID: 15915565 [PubMed - indexed for MEDLINE]
Thanks for that David. I have used a similar auto-induction medium in the past – they are very good.