Have you met my new 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 👉 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 👉 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 👉 Making mRNA copies of the gene (TRANSCRIPTION) & 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 causes the mRNA to fall off & frees T7 Pol gets freed to make more copies! 👍

And you want to make LOTS of copies of the mRNA bc this DOES have to compete w/bacterial mRNA for the bacteria’s protein-making machinery (RIBOSOMES) 😬 BUT bc 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 👉 devote almost all their resources to expressing our gene 😏 after just a few hours >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 👉 can’t reproduce any more 😩 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 😬

1 way to do this is to just not give it T7 Pol. And in fact, if you 👀 at a pET vector like Hector you’ll see it does NOT have the T7 Pol gene 👉 Without this, your gene will lay dormant 😴 We rely on the bacterial host DNA and NOT the plasmid DNA to provide T7 Pol 👍Bacteria don’t normally have this gene, 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 👉 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 👉 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 😬 👉 can’t fold our protein properly ⏩ protein forms clumps of aggregates called inclusion bodies 😬 BUT we can ⬇️ expression by ⬇️ inducer concentration (add less IPTG) 😅

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! 👍

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