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

[@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!!!

[@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

[@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).

[@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?

[@ladidakalana2202]

Such an informative video!kudos to youu girl

[@thebumblingbiochemist]

Thanks

[@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.

[@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

[@Chesonate]

Thank youuuuu!

[@thebumblingbiochemist]

Happy I could help!