How to Shut Off Background Lac Expression in LB

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.