Re: [DIYbio] Re: Promoter Testing

Nathan brings up a good point. Do you want a comparison between different promoters or an absolute measure of expression rate? Comparisons are much simpler so long as you control your experiment well, run multiple repeats in tandem, and use the same media batch throughout. Using different strains would be interesting too and can back up your claim that promoters produce the same amount of protein across the board. Just go with a popular strong constitutive promoter as your standard and compare all to that.

Then repeat the whole experiment a few more times for statistical strength and plot all data with error bars. If you really want to go all-in on this experiment you can look up ways to calculate the sample size needed for your specific confidence and the effect size. You can show very high P values stating the means are not alike but how much they are not alike is more of an indicator of strength. The how-much would come from the effect size. Note that Cohen's d (effect size) is generalized for behavior biology and a recent navy or army analysis says using those standards are misleading. Meaning an effect size of 0.2 which Cohen deemed as "small" may be quite large for the study at hand when the results of other similar experiments show a much smaller range of possible values thus making it large.

Again, if this is just a one-off experiment for fun and curiosity you don't need to do any statistics but I strongly encourage you to give it a shot since the experiment is simple enough that some repetitions wont be too painstaking. To quote my friend who just texted me a science joke:

"To consult the statistician after an experiment is finished is often merely to ask him to conduct a post mortem examination. He can perhaps say what the experiment died of"

Sebastian S. Cocioba
CEO & Founder
New York Botanics, LLC


> On May 14, 2015, at 10:20 PM, Nathan McCorkle <nmz787@gmail.com> wrote:
>
>> On Thu, May 14, 2015 at 5:42 PM, Linden <ff.lindenberg@gmail.com> wrote:
>> Sure, I dont have it with me right know, but it was something like this:
>
> So does that mean you only tested a single promoter? It would really
> make more sense and be easier if you grew up multiple strains in the
> same manner, with different promoters, then compared them to each
> other. Otherwise, you'll need to look up published data and compare
> using their methods.
>
> You could also purify the GFP with, either ion-exchange chromatography
> columns (pretty cheap and easy), or HPLC, etc... then you know the
> elution volume, but not the concentration... but since it should be
> pretty 'pure' you can compare to published fluorescence values to get
> the concentration. When you know the concentration and the starting
> volume of GFP cell culture, along with the cell-density of the
> solution, you can get a value for GFP per cell.
>
> If you really just want to compare promoters, either do what I said
> and test them side-by-side... otherwise you need to replicate
> previously published methods otherwise comparing to them won't make
> sense.
>
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