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How to Shut Off Background Lac Expression in LB

lac expression

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- hydrolyzable analog 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 metabolizable 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.

Originally published on April 22, 2008.  Updated and revised on March 31, 2016.

lac expression

3 Comments

  1. Shyam Bhakta on October 19, 2018 at 8:00 am

    Who uses the wild-type lacZ promoter any more? It’s more common to use a synthetic/hybrid LacI-repressed promoter that doesn’t have lacZp’s Crp (CAP) or H-NS operators, in which case glucose won’t inactivate the promoter as there’s no Crp sites positioned to induce expression with CRP:cAMP. Not even pET vectors have a CRP site in the T7 promoter (T7 RNAP can’t even be activated by CRP, which interfaces with the α subunits of E. coli RNAP).

    I imagine a non-milk-based tryptone or peptone for the nitrogen source would work to eliminate the lactose.

    • Dr Nick Oswald on October 19, 2018 at 12:35 pm

      Plenty of legacy plasmids use the wild type promoter. For those, the solutions in the article are ideal.

      That said, your point about using variant promoters to eliminate the problem is a very good one. Worthy of their own article 😉

  2. IsaacChavez on September 3, 2014 at 9:52 pm

    Do you have any evidence?

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