I have got transformed Rosette gami cells. I have used pET vector containing histone protein insert. If I seed these transformed cells on IPTG incorporated LB agar plates, will the cells start expressing histone proteins on plate itself?
It depends on the IPTG concentration... If it's too high, it will inhibit growth because the cells will shift totally to protein production. But it's possible to express protein in plate, I've done it before.
It depends on the IPTG concentration... If it's too high, it will inhibit growth because the cells will shift totally to protein production. But it's possible to express protein in plate, I've done it before.
In liquid culture, growth rates go down severely when protein expression is induced. At least this is what I observed. Whe you add IPTG to the plate medium the metabolic load on the Colis will definitely cause the colonies to grow much slower.
As Joao says, a low IPTG concentration should be the best start.
By the way, does anybody have a reference for the kinetics of the IPTG inducible T7lac promoter? All I could observe until now is that the induction level as a function of IPTG concentration is not linear...
Naveen> Yes they will, but you will also get lots of plasmidless cells, rearranged/mutated plasmids and so on, so you might want to be careful with what you use them for. Agar plates are different from liquid cultures, as there are antibiotic concentration gradients, higher competition for nutrients, etc. and non-expressing cells will enjoy a significant advantage. You can take a look here for an idea of what may happen when protein expression is not repressed: http://www.microbialcellfactories.com/content/8/1/8
For sure E. coli will start to express the protein, when the medium (also on plates) contain a suitable concentration of the inducer (in your case IPTG). This was done several times before, e.g. in high throughput screening or selection systems.
Xenoprotein expression in E.coli is not always as simple as it in theory. Several factors affect the expression such as IPTG conc, temperature, nature of protein, whether it is tagged or not and so on. In any case, if it works at RT or standard 37C at 1mM IPTG conc, i wonder what use it will serve on plate for you......
Something that we sometimes find in my lab is that expression of a heterologous protein during sustained growth on solid medium (i.e. sufficient number of cell divisions to form a colony) can become toxic to those cells. If so, one way around it is to use standard ZYP-5052 auto-induction media with 1.5% (w/v) agar added to it to form solid plates. On such media, expression of the protein will not be induced until the colony has already reached a decent size. If interested, this medium is described in detail in one of our papers: Owen et al. 2011. Rapid and flexible biochemical assays for evaluating 4'-phosphopantetheinyl transferase activity. Biochemical Journal 436: 709-717.
Dear Coelho, probably you missed to read IF in my post. I was just giving a hypothetical condition among n number of possibilities. Never mind.. cheers....
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