have anyone ever encountered a situation that small scale expression (0.3 ml 96 well plate) is not reproducible when repeated in 500ml flask?
the conditions of both expression is the same, i.e: T=30 degree Celcius, rpm=200 rpm except as listed below:
96 well plate:
a) culture volume: 0.2ml culture
b) aeration: 2/3 (ratio culture volume to well volume)
c) post-induction time: 1 hr after inoculation
flask:
a) culture volume: 100 ml culture
b) aeration: 1/5 (ratio culture volume to flask)
c) post-induction time: at OD600 = 0.7
the relative expression in 96 well and flask are slightly different. For 96 well, it increased 4 -fold compared to WT but the same expression increased only 2-fold in flask.
can anyone suggest me relevant reference paper on this?
1. Where the WT grew in? Did you grow WT in 96-well and flask for 96-wll and flask controls respectively?
2. The aeration spaces (affect oxygen supply) were different for 96-well and flask, this could affect the experimental results.
3. Although the shaking speed (220 rpm) was the same, the agitation effect could be different for cells in 96-wells and cells in flasks.
As already said before, your conditions are not the same in your 96 well plate and your shaking flask. You should ask yourself why the 96 well plate works better, e.g. do the cells grow comparable in both cultivation devices (same growth rates, oxygen limitation etc.)? What is the influence of the start and end of the expression phase (in your 96 well plate you induce with IPTG rather early)? What is your inoculation density? Did you use the optimized clones from your plates for the flasks and how did you do this (my advice: prepare a copy of your 96 well plate, that you use for glycerol stocks that is not induced with IPTG)? And finally, at least you see the same tendency (increase) in both cultivation systems - this could be worse and I had several trials in which the upscaling failed completely...
Abbas,
As Yuan noted, above, you are actually probably aerating at a significantly higher rate in your flasks than the multi-well plate. Since your results on your plate were what you desired, I suggest taking a small amount of dye (coomassie for gel staining which you likely have on hand would work well, and perform parallel experiments: 1) figure out how much dye you need to use to be able to accurately detect even dissipation in both a well, and in a flask. (hopefully the overall volumetric ratio will be similar, but perhaps you will need a little more dye/volume in the well due to the shorter path length); 2) using a pipettor (or micropipettor for the plate) drop a small amount of dye into the desired volume of diluent in your well (parallel experiment in the flask) do not stir, but begin agitation and track how long it takes for the dye to be evenly distributed in your multi-well plate versus the flask. Your aeration and degree of mixing will be more closely matched in the flask when the cycles/second for your flask mixing gives you about the same dye dissipation time as in your multi-well plate.
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