Quick note from the Bibel lab 1 - autoinduction pt. 1

Auto-induction is an alternative to IPTG-based induction for genes under regulation by a lac promoter. The media involves a mix of glucose and lactose. The bacteria prefer the glucose, so they eat that first, but when that runs out, they switch to lactose. They use their lac promoter to control the switch (switching on lactose metabolism genes only when there's low glucose and high lactose so they don't waste resources making stuff they don't need). If we stick another copy of the lac promoter in front of a gene for making our protein of interest, the switch will also turn on the expression of our gene. In the traditional IPTG method, you monitor bacterial growth (via OD600 measurement), then add the allolactose mimic IPTG once the bacteria are at a high density (so there's lots of bacteria able to devote themselves to making your protein), but not overgrown (so the bacteria aren't dying and releasing proteases, etc. when you ask them for favors).
blog: tinyurl.com/8esuevm7
Then, once the OD600 is where you want it, you add the IPTG, which tricks the bacteria into thinking there's lactose around. The lac promoter is derepressed, allowing for translation of your gene of interest*. In the auto-induction method, the bacteria naturally grow to a good density in the presence of glucose. But then, when the glucose runs out, they plateau during the switch to lactose. During this time, the lac promoter is derepressed, and translation of the lac operon and your gene of interest starts up. No monitoring required!
*often you put the production of T7 polymerase under lac promoter control, and then your protein of real interest under control of T7 promoter (and sometimes also lac, with hybrid T7lac promoters for extra tight control)
Most autoinduction protocols use a defined media, where all the components are known exactly and precisely controlled. But that means you have to measure, combine, etc., a lot of different components. And then make high concentration stock solutions that are a pain to dissolve (yes, I know this first-hand). Finished below…
Based on Studier, 2005: Studier F. W. (2005). Protein production by auto-induction in high density shaking cultures. Protein expression and purification, 41(1), 207-234. doi.org/10.1016/j.pep.2005.01...
So I was really excited to get this simplified protocol from Walter R.P. Novak, Ph.D. William J. and Wilma M. Haines Professor of Chemistry Wabash College.
What makes it a lot simpler is that it uses a “complex” media as a base (to which glucose & lactose are added). Despite the “complex” in the name, it’s actually much simpler to make. The “complex” just refers to the fact that we don’t know its exact composition (which might vary from batch to batch, making it less reliable). One way to find out though! If it doesn’t work out, we’ll try alternatives, and go back to the conventional IPTG method if needed. But, since we’ve got other things going on simultaneously, this seemed like a great first approach.
This also serves as the start of my test runs for next semester - I’m hoping this will simplify expression of MDH (malate dehydrogenase) greatly in my lab courses next semester!
I will hopefully provide an update at the end of the week about how it goes/went!
IT WORKED!
Here’s the protocol from Dr. Novak:
For 800 mL of media:
Tryptone: 16 g
Yeast extract: 4 g
NaCl: 4 g
Autoclave, the above then add (we’re trying premixing these):
(All are sterile filtered)
60% glycerol (v/v): 8 mL
10% glucose (w/v): 4 mL
8% D-lactose (w/v): 20 mL
To use, grow a 5 mL overnight culture at 37 ˚C in LB media
Inoculate 800 mL of autoinduction media supplemented with the appropriate antibiotics with 4 mL of overnight culture (1:200 dilution). Grow for 24 hours at 30 ˚C. Harvest! I think some people grow at 37 ˚C also, but I would probably try for a shorter growth time; however, it is important to have the cells run out of glucose to use the lactose :)
And here are recipes for making the traditional Studier media
pro.unibz.it/staff2/sbenini/do...
P.S. I’m pretty sure 5052 = 5% glycerol, 0.05% glucose, 2% lactose (w/v speaking)
Also, I’ve heard from numerous people that either they don’t add the metal mix or just use the iron
More about IPTG-based induction: bit.ly/bacterialproteinoverexp... & • IPTG induction using t...
For images and more: tinyurl.com/mt5n5rh4
more about all sorts of things: #365DaysOfScience All (with topics listed) 👉 bit.ly/2OllAB0 or search blog: thebumblingbiochemist.com
#scicomm #biochemistry #molecularbiology #biology #sciencelife #science #realtimechem

[@huchang9794]

nice to see you again😁

[@steveholmes1736]

I just love this girl! Another well described molecular biology video.

[@thebumblingbiochemist]

Thanks! Glad you enjoyed it! There are accompanying figures in the blog version tinyurl.com/33dj6efy

[@ourworld6237]

😂 I like how your always smiling

[@thebumblingbiochemist]

Hard not too when you're so fortunate