Hi Everybody,

We are looking into using AutoInduction in out HT expression in E.coli. The plan is to use it both in our small scale screen, (96 deep Well blocks) and in large scale expression (0.7 - 4.5 L) Shake or bubble flasks.

Do any of you have experience with autoinduction in these settings?

All inputs are very welcome.

Cheers

Bjørn

We use auotinduction in small scale (24well), 50-100ml flask scale, 1L and 10L  high cell density scale(HCD). oD600 are typically 10-20 in 24 well, 20-40 in HCD fermentation depending on the feeding.It works perfectly in all settings in a scalable manner. However, for HCD Lactose + YE fed-batch seems better than batch, but often we do just batch.We compared the Studier Recipe with Novagen. Expression levels were equal, optical densities in 24 well some times better with Novagen, they may have included some more goodies.

we use auto-induction at all scale (1 ml-10 litre, no fermentation here) for years,  We used them side by side with 2YT, TB, SB, LB at some point and dropped the other medium since. We published our protocols/data this year if you are interrested (Methods journal). If you use DW24 you get the same OD than in shake flasks (as Sabine mentioned 10-20 depending on temperature) in a normal shaker BUT if you want to be sure to get reproductible growth and results in DW 96 than in flasks you should use small orbital shakers (we use Infors 0,5 mm orbital at 900 rpm).

Renaud

Thanks Renauld and Sabine,

This is very helpfull information, we will try Renaulds protocol with a variety of different proteins to see if it works in our hands.

I'll be back with the results when we have them.

Best

Bjørn

Hi,

I met several people that were not as happy as us with ZYP5052. In many cases they were making the same mistake which is to start in the morning and harvest in the evening 6-7 h post dilution of the preculture like they would do for LB for instance (which would be 3-4 h post induction with LB). That's far too early for ZYP at 37°C where the cultures are still in growth phase and harvesting there make you loose most of the benefits (induction starts not before 2-3 and can be as late as 10 to start with good aeration).

So be patient and don't harvest cultures with less than 10-12 OD in any case. When we work on hundreds of cultures, we sometimes have few cultures with OD between OD 4-10 (preculture not saturated for instance), they often work and we know that if we grow them higher we will have more proteins  in most cases.

Renaud

Thanks for the tip,

We can certainly let them grow longer than 6-7 hours, our std is to let them express ON.

I think that our first approach will be the 4h at 37 then downshift to 18. In your experience, do you reach the same OD's at the lower temperature?

Thx

Bjørn

"I think that our first approach will be the 4h at 37 then downshift to 18. In your experience, do you reach the same OD's at the lower temperature?"

Hi Bjorn,

we get between 10-12 in the morning at harvesting time, better 12 with lots of soluble than 20 with lots of insoluble (yes, we would probably sometimes have lots of soluble with OD 20 as well...).

Fusion tags and temperature: when working with fusion tags (TRX/MBP...) in our default setting at 37/17 we sometimes see 2 bands (I'm sure everybody have seen this:-), the fusion alone and the Fusion-ORF, in a lot of cases growing 2 days at 17°C solves the problem and we get only the Fusion-ORF protein for the dowstream process.

I never moved out of ZYP5052 because initially Bill gave us the receipe of this one alone (receipe from 2002), does anyone has experience with ZYM-5052 versus ZYP5052?

Renaud

Hi all!
At PEGS Europe last year, there was a conference where a group developed a autoinduction minimal medium that seemed to work fine from small-scale to large-scale. The details of this medium can be found in:

Appl Microbiol Biotechnol (2011) 92:823–833 

Appl Microbiol Biotechnol (2011) 91:1203–1213

We are now trying it, so I can't give any feedback on our experience with it!

Hope this helps!

Dear Bjørn,

I'm a bit late for this discussion, but in case you're still interested in suggestions for autoinduction media, I would like to contribute one more.

One postdoc at our institute used TB-FB medium plus ~1.5% lactose for her culture and got good expression yields.

TB-FB is prepared as following:

1 L of TB-FB = 900 ml of Extract stock + 100 ml of Phosphate stock

in 900 ml Extract stock: 12 g Bacto-Tryptone, 24 g Yeast extract, 4 ml glycerol, pH 7.2

in 100 ml Phosphate stock: 2.314 g KH2PO4 (=0.17M), 16.43 g K2HPO4*3H2O (0.72M)

Here's the workflow with the "MJ medium" (named after her name, Mandy Jeske):

preculture in LB medium 100 ml overnight, 37°C

500 ml TB-FB medium + 37.5 ml 20% lactose + 25 ml pre-culture

24-26 hours @ 22-25 °C

final OD 20-30 (construct dependent)

I hope that works for your setup as well.

Best,

Hüseyin