Shake it while you make it – and make it starting… now! Even though these flasks may be (more than) half empty, now IPTG’s been added to induce overexpression, the bacterial cells in them will hopefully soon be half full with my protein! Thank goodness for recombinant protein expression letting us use bacteria to make lots of proteins of our choosing on demand! I mean, seriously!
I’m reading this “For the Love of Enzymes” book by Arthur Kornberg – he won the Nobel Prize for discovering the DNA copying machinery, DNA Polymerase (DNA Pol). He was able to isolate DNA Pol from bacteria & use it to make copies of DNA in a test tube – something we now take for granted all the time in the lab when we do PCR – but which was a major breakthrough. And took a LOT of hard work – first by the bacteria to make it – and then by the scientists to purify it.
Cells make a lot of different proteins (like, literally thousands), and DNA Pol is just one of them. This was at the beginning of the “molecular biology revolution,” before scientists became able to mix and match (recombine) pieces of DNA to better control & manipulate them. So he had to isolate DNA Pol from the cells’ normal levels of it – and normally they don’t need to make that much of it. So he’d need a lot of cells – how many? He had to use a giant (10,000 L) fermentation vat!!!!! (usually used for alcohol making). This got them 200 pounds of bacteria (~200 KILOgrams) and, after about a month of hard work, roughly 1/2 of a gram of purified DNA Pol!
Nowadays, we can stick the genetic instructions for a protein we want made into bacteria and get the bacteria to devote almost all their time & resources into making that one specific protein – so instead of 10,000L, today I’m expressing 8L (and I’m only going to purify 1 or 2 at a time, I just want “backups” because I’m trying to optimize my purification scheme). Not only can I tell them to make 1 protein – I get to tell them exactly what protein I want made – and when I want it made
I do it with the help of my pET Hector. He’s a VECTOR! He can’t play fetch but he *can* be used to make PROTEIN! & he can do it on command! This might come as a shock but “Hector” is just a nickname, His given name is pET28a(+). pET stands for *p*lasmid *E*xpression vector under *T*7 control. It’s a family of plasmids that’s 1 of our go-to tools for RECOMBINANT PROTEIN EXPRESSION in BACTERIA. This is where we stick a gene with instructions for a protein into a piece of DNA called a PLASMID ⭕️ ➡️ stick that plasmid in bacteria ➡️ have that bacteria make our protein & it works by taking a page (& genes) out of T7 PHAGE’s book
T7 is a bacteriophage or “phage” (a virus that infects bacteria) & it has a simple goal: reproduce! T7 has a small, linear, DNA genome (complete genetic blueprint more making identical copies of itself)). It coats itself in a protein shell when it travels, then latches onto bacteria & injects its DNA into it, The bacteria copy this DNA for them & use it to make the proteins it needs to coat itself & inject into other bacteria 💉
They have to convince the bacteria to make *their* proteins instead of their own bacterial proteins. And this is what we want to do too!. So we learn from (*steal from*) the masters. So how do they do it? It’s all about bypassing the holdups: there are 2 main “holdups” that can get in their way 1) Making mRNA copies of the gene (TRANSCRIPTION) & 2) making protein from those mRNA copies (TRANSLATION)
TRANSCRIPTION (DNA➡️RNA) requires an RNA POLYMERASE (RNA Pol). Bacteria have their own, but it’s busy making bacterial proteins, so rather than rely on the bacterial RNA Pol, T7 makes its own RNA Pol (T7 Pol). And this one is specific for its own PROMOTER (start site on the DNA the Pol latches onto). So T7 gets this one all to itself – the bacteria can’t use it.
The PROMOTER tells T7 Pol where to START but how does it know where to stop? The T7 TERMINATOR! This is a sequence that, when copied, folds into a hairpin which causes the mRNA to fall off & frees T7 Pol to make more copies!
And you want to make LOTS of copies of the mRNA because this DOES have to compete w/bacterial mRNA for the bacteria’s protein-making machinery (RIBOSOMES). BUT because T7’s so active & exclusive it can easily swamp out the bacterial mRNA.
Similarly, if we put a T7 PROMOTER before our gene, a T7 TERMINATOR after it & give it some T7 Pol, we can get bacteria to OVEREXPRESS our protein. With bacterial overexpression, you get the bacteria to devote almost all their resources to expressing our gene – after just a few hours over ½ of all protein in the cell could be ours 🤯
BUT because the bacterial cells are devoting themselves to making our protein, they’re neglecting their own needs – including reproduction – that reason why bacteria are so useful in the lab (well, one of many reasons) is that their population booms rapidly because it doesn’t take them long to copy all their DNA (DNA replication) then split in half, giving each new cell a copy. That takes a lot of energy and resources, which the bacteria doesn’t have if it’s devoting itself to T7 protein-making. T7 doesn’t care about this, but we *do*, bc we need to be able to grow the cells to get enough cells to express lots of our protein
One way to do this is to just not give it T7 Pol – that special polymerase that makes the RNA copies of the T7 genes (which ribosomes use to make T7 proteins) or anything that “looks” like a T7 gene because it’s under the control of a T7 promoter (like the gene we want to express). And in fact, if you look at a pET vector like Hector you’ll see it does NOT have the T7 Pol gene. So how does our protein get made? Wasn’t the whole point of using the T7 promoter to make a lot of it?! Don’t worry – we still have the T7 Pol gene – we just keep it separate so we can activate it “on command”
We rely on the bacterial host DNA and NOT the plasmid DNA to provide T7 Pol. Bacteria don’t normally have this gene (it’s from a virus that wants to sabotage it, remember), but specific strains of bacteria have been designed so they DO. If we’re still in the cloning phase & only want to make more copies of the plasmid ⭕️➡️⭕️⭕️⭕️ we can stick it in bacteria that don’t have it (strains like DH5α). And then, when we want to express it we stick it into bacteria that DO have it (like BL21(DE3))
BUT we still want more control – we want to be able to control when those bacteria that *have* the T7 Pol gene actually *make* T7 Pol. So we steal from another clever biological setup – the LAC OPERON, to be able to control *when* we express the protein ⏰
Bacteria use the LAC OPERON to control when they make the machinery for breaking down the sugar lactose. More here: http://bit.ly/2MxNPs2
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 ⏩ repressor changes shape & falls off, freeing the promoter for RNA Pol binding
If we stick a lac promoter in front of the T7 Pol gene & don’t give the bacteria lactose (it’d rather eat glucose anyway) the T7 promoter will stay hidden, so no T7 Pol made. For tighter control, we can stick one of these lac promoters in front of the T7 promoter in front of our gene as well, giving us a “T7lac” promoter
When we add the allolactose mimic IPTG (Isopropyl β-D-1-thiogalactopyranoside) it binds the repressor ⏩ repressor falls off ⏩ bacteria makes T7 Pol ⏩ T7 Pol binds T7 promoter in front of our gene ⏩ T7 copies the DNA into RNA until it reaches the T7 terminator & they come apart ⏩ 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
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. BUT we can lower expression by reducing inducer concentration (add less IPTG) and/or growing at a lower temperature
Moral of the story 👉 although pET VECTOR’s designed for expression, you need to make sure the cells you put it in are too if expression’s your goal! But if bacteria don’t work, all hope’s not lost! 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 express a lot of my proteins using those, and you can learn more about how here:http://bit.ly/2SLCt6N
But when I can use bacteria, I do because it’s way cheaper & easier – and – when it works – you can get a lot more protein per liter. They have really simple growth conditions – they grow fastest at ~37°C, so we set the shaker incubator thermostat to this nice warm temp when we want them to grow and multiply lots. The shaking is important because it makes sure the cells stay aerated – each cell gets a chance to be closer to the oxygen and CO2 doesn’t build up – for proper aeration you need to leave a lot of empty space in the flask (like at least 3/4 of what the flask says it holds). I do small “starter cultures” overnight (50ml) so I can get a lot of cells to start with. Then I add some (usually ~5ml) to 1L portions of media in 4L flasks.
Now I have to start monitoring its growth – I want them to grow enough that I get lots of cells (my “factories”) but I need to make sure each of these factories gets enough supplies & doesn’t have to compete with one another for resources. So I periodically check the OD600 to tell me how dense the media is which (the more cells there are the harder it is for light to pass through it) and we can measure this cloudiness as the “Optical Density” measured by a spectrophotometer that shines light (in this case light with a wavelength of 600nm) through a sample of it in a little square “tube” with clear walls called a cuvette and measures how much of the light makes it through. http://bit.ly/2KZ8vKQ
What’s the optimal optical density for induction? It’s protein – and media – dependent. For LB (Lysogeny Broth) I normally aim for ~0D 0.6-0.8. TB media is more nutrient rich, so it can support denser cell growth – I usually aim for an OD600 of ~1.4-1.8. Once I see it getting close, I move the flasks to the cold room and decrease the incubator temperature to 16 or 18°C.
I typically add IPTG to 1mM but the optimal amount is protein-dependent once again. When I add IPTG, T7 Pol gets made. So my T7-promoter-controlled gene gets copied into mRNA. And then the ribosomes start making protein from it. I let them make protein overnight at that 18°C temp – at this lower temp protein making’s slower which gives proteins more time to fold the right way and hopefully prevent aggregation.
In the morning, I can “harvest” the cells by pouring the liquid holding them into bottles, centrifuging them (spinning really fast to pellet them out cuz they’re heavier than the liquid), re-suspending them in a bath of nice clean buffer (pH-stabilized salt water), then breaking them open (lysis) and purifying out my protein – which is made “easy” because I’ve used DNA Pol to help me redesign the gene to add a little tag onto the end that will specifically bind little beads (resin) in affinity chromatography. http://bit.ly/2XBu9eI
Kornberg clearly didn’t have this luxury either – to purify it the first time he had to go by “trial and error” – changing salt conditions to separate whatever was in naturally there by their solubility (when the solution became too salty for various proteins they’d “crash out” and precipitate). And then he’d test whether the DNA-copying activity was in the precipitate or the remaining liquid (supernatant) and then keep going, chasing down something he didn’t even know what was – just by following the activity – pretty freakin amazing if you ask the bumbling biochemist! (He did his trial-and-erroring on smaller volumes to establish a protocol before scaling up to that massive vat!)
So yeah – I’m feeling pretty grateful! (for recombinant protein expression and so much more!)
more on types of growth media: http://bit.ly/bacterialmedia
more on different expression systems: http://bit.ly/2G5N5tY