IPTG induction using the lac promoter

The basic principle behind recombinant protein expression is that we can stick the genetic instructions for a protein we want made into cells from a different organism and it’ll make the protein for us. We can steal a clever biological setup from a bacteria-infecting virus (a phage) - the LAC OPERON, to be able to control when we express the protein, so we can allow the bacteria to grow lots before we get them to focus on protein-making and not dividing. All we have to do is stick a lac promoter in front of our gene of interest, then add IPTG when we want the cells to make protein. This tricks them into thinking lactose is present and they need to make lactose breakdown machinery.

Much more detail here: http://bit.ly/bacterialproteinoverexp...    ; longer video with more on the T7 system: Inducible protein overexpression in b...  

But here’s the just the gist version… IPTG-inducible protein expression usi...

A gene (DNA recipe for making a protein)’s “natural home” is in a chromosome, which is a really long strand of DNA that holds a lot of genes. We’re only interested in one (and we want the edited version - the DNA copy of the mature mRNA, which we call complementary DNA (cDNA). So, using methods like “cutting and pasting” with restriction enzymes and DNA ligase or “copying and stapling” using PCR-based methods like SLIC, we can stick the cDNA for the protein we want made into a smaller piece of DNA that’s easier to work with and which has some special features. We call this carrier DNA a “vector” and for bacteria, the vector is usually a small circular piece of DNA called a plasmid.  
 
Bacteria use the LAC OPERON to control when they make the machinery for breaking down the sugar lactose.  They only want to make that machinery if there’s lactose present, so when there isn’t, a repressor protein (LAC REPRESSOR) sits on the LAC PROMOTER site where RNA Pol needs to bind & “hides it” Then, when lactose is available, some of that lactose gets converted to allolactose which binds the repressor. This causes the repressor to change shape & fall off, freeing the promoter for RNA Pol binding  
 
If we stick a lac promoter in front of our gene & don’t give the bacteria lactose (it’d rather eat glucose anyway) the T7 promoter will stay hidden, so none of our protein will be made. I say “no” but the promoter can “leak” if the repressor falls off on its own and RNA Pol sneaks in before it rebinds. So, for tighter control, we can stick do things like use the lac promoter to control expression of T7 Pol & use a T7 promoter in front of our gene of interest.

Instead of adding lactose or allolactose, which the bacteria can break down, we add the allolactose mimic IPTG (Isopropyl β-D-1-thiogalactopyranoside), it binds the repressor ⏩ repressor falls off ⏩ RNA Pol binds promoter in front of our gene ⏩ RNA Pol copies the DNA into RNA ⏩ does this over & over 🔁 making lots of mRNA copies that swamp out the bacterial mRNA & outcompete for the limited ribosomes ⏩ ribosomes make our protein from the mRNA instructions ⏩ we celebrate! 
 
Well, sometimes we celebrate. But sometimes we’re not so cheery because sometimes they make too much for the cell to handle, so the cell can’t fold our protein properly & the protein forms clumps of aggregates called inclusion bodies, and when we break open the cells (lyse them) to get out our protein and then spin them down (centrifuge them) to pellet out the insoluble stuff like membrane bits, and we expect our protein to be in the liquid part, its actually with a bunch of crud in the pellet. BUT, all hope’s not lost - we can try again & lower expression by reducing inducer concentration (add less IPTG) and/or growing at a lower temperature. 
 
But sometimes that’s not enough to get you the protein you want. It’s easiest to explain recombinant protein expression in terms of bacterial expression systems, and a lot of proteins are expressed this way (probably most of them) - but some proteins don’t express well (or at least they don’t survive the expression process well) in bacteria - because even though bacteria have all the copying machinery, they don’t have the same folding helpers and post-translational modifiers our cells do - so they can misfold & clump up, have different phosphorylation (added phosphates) & glycosylation (added sugar chains) patterns 
 
So for these trickier proteins we can express them in cells more like ours - mammalian cells are harder (but doable), but insect cells like Sf9 aren’t too bad. I expressed a lot of my proteins using those in the past. finished in comments