Recombinant protein expression in bacteria using the inducible T7/pET system

Want a lot of protein made on demand? And you don’t just want it expressed - you want it over-expressed? Take a page out of the book of T7-phage! In recombinant protein over-expression in bacteria, I can stick the genetic instructions for a protein I’m interested in into a plasmid vector (like pET28) and get the bacteria to devote almost all their time & resources into making that one specific protein - I get to tell them exactly what protein I want made - and when I want it made, through IPTG induction of T7 polymerase. ⠀ 
 
blog form w/full text (also has static graphics): bit.ly/bacteria...  
 
pET stands for *p*lasmid *E*xpression vector under *T*7 control, and it’s a family of “phagemid” vectors that mix and match features of plasmids: small, circular DNA that can survive and get copied even though they’re “extrachromosomal” (not part of the bacteria’s own chromosome); and bacteriophages or “phages” (a virus that infects bacteria). One such phage is T7, and, like other phages, it has a simple goal: reproduce! ⠀ 
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They have to convince the bacteria to make their proteins instead of their own bacterial proteins. And, for recombinant expression, 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)⠀ 
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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 to start making recipe copies). So T7 gets this one all to itself - the bacteria can’t use it.⠀ 
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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! ⠀ 
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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.⠀ 
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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 your gene - after just a few hours over ½ of all protein in the cell could be yours ⠀ 
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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*, because we need to be able to grow the cells to get enough cells to express lots of our protein ⠀ 
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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, 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”⠀ 
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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.
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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
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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 ⏩ we celebrate!⠀ 
 
more on troubleshooting: bit.ly/wheresth...

[@dangthatscool1]

So glad to find your channel --- this is exactly what I need and at the level I need it! Thank you for your efforts!

[@thebumblingbiochemist]

Thank you! So glad I could help

[@Sheebert]

Thank you for this video. I’m going to be researching specific proteins this summer, and needed a refresher on overexpression!

[@thebumblingbiochemist]

Good luck!!!

[@exciton007]

I love your videos. You are such an intelligent, inspiring young woman. Thanks a lot!

[@thebumblingbiochemist]

Thanks so much!

[@sherdilasif4143]

Hi, brilliant video, I am purifiying a single chain fragment antibody and I am wondering why my plasmid has a lac promoter instead of the T7 promoter? its a pkamp pET plasmid

[@thebumblingbiochemist]

Thanks! I'm not familiar with that particular plasmid but the lac promoter typically controls the production of T7 and then your gene of interest has a T7 promoter. Sometimes, the gene you’re expressing from the T7 promoter will also have a lac promoter in front of them - this makes expression even tighter, so you get less “leaky expression” from small amounts of T7 that get made even without induction (i.e. T7 that is constitutively expressed at low levels).

[@ladidakalana2202]

Such an informative video!kudos to youu girl

[@thebumblingbiochemist]

Thanks

[@michelleyoonjiamin5165]

Hi Professor Bri, not sure if you will be looking at this comment but would like to ask a question with regards to protein expression (via IPTG) using BL21(DE3) E. coli cells. Have you encountered a situation whereby uninduced BL21(DE3) E. coli producing the protein of interest with quantity that is comparable to IPTG-induced BL21(DE3) cells? Is there a possible explanation to what I am experiencing now? It is definitely beyond leaky expression as leaky expression would normally give a faint and thin band on the SDS-PAGE (compared to induced cells). Thanks in advance!

[@thebumblingbiochemist]

Hi - have you verified that the band you're looking at is actually your protein of interest?

[@hitkarshkushwaha2434]

Great!

[@hazemdarwish3659]

So, you centrifuge the bacteria and resuspend in a smaller volume of buffer containing PI and flash freeze using liquid nitrogen and store in -80C, then lyse these cells later by whatever method. I wanted to ask you from your experience, would it matter if i have cultire pellets ( about 10 grams) and just freeze them in -80C as they are in solid form with no solution? also would they need a washing step prior to being frozen in that state (solid form) ?

[@thebumblingbiochemist]

You can do either and different people have different preferences but I don't know how much it actually affects the results. Typically I don't wash the bacterial pellets.

[@Chesonate]

Thank youuuuu!

[@thebumblingbiochemist]

Happy I could help!

[@msteriouscm302]

I'm curious, is there any recommended stock concentration for amino acids?

[@thebumblingbiochemist]

for what purpose?

[@msteriouscm302]

@@thebumblingbiochemist I just want to prepare for reviving of my sample and checking their aa requirements.

[@thebumblingbiochemist]

I still am not sure what you're trying to do or asking sorry