Promoter Testing

[Linden]

Hi guys!

I would like to rank in strength a few promoters I have. The promoters are cloned upstream GFP, so I used a spectrophotometer (485 nm excitation 525 nm emission) to measure the GFP in E. coli, just like the paper "Measuring the activity of BioBrick promoters using an in vivo reference standard". I did everything, and now I have a some numbers, but I don't know how to turn it in something useful. Anyone know which formula should I use to get a estimate of the GFP or mRNA concentration inside the cell?

Thanks a lot!

[Nathan McCorkle]

Can you post the data? How does the data look when you sort by
emission counts then plot it?

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-Nathan

[Linden]

Sure, I dont have it with me right know, but it was something like this:

I inoculated colonies in LB and let them grow overnight. In the morning I diluted it in M9 medium and let it grow for 4 hours, then I did the first measurement (T0), I measured the OD and fluorescence. After another 1 hour of incubation I measured the OD and fluorescence again (T1).

At T0 the fluorescence was around 50 and at T1 it was around 70, the OD I cant remember. It would be great to get an estimate of the GFP concentration inside the cell.

Thanks!

[Sebastian S Cocioba]

You may need a gfp standard curve to know for sure. Anything else would be back of the envelope. Would it be feasible to buy an isolate of your fluorophore or hisTag it and purify? Thete should be papers on measuring this but IMHO taking optical density readings of bacteria in stationary phase against a fluorescent protein standard curve using YOUR spectrophotometer (not all created equal) would be the most quantifiable.

Sebastian S. Cocioba

CEO & Founder

New York Botanics, LLC

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[Nathan McCorkle]

On Thu, May 14, 2015 at 5:42 PM, Linden <ff.lin...@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.

[Sebastian S Cocioba]

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

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[Linden]

Hey guys,

Thanks Nathan and Sebastian, I will compare the promoters. Just to clear it out, there is no way to know the amount of GFP inside the cell using only the spectrophotometer results? Lambert-beer equation doesn't apply in this case?

On Thursday, May 14, 2015 at 8:02:45 PM UTC-3, Linden wrote:

[Nathan McCorkle]

On Fri, May 15, 2015 at 10:09 AM, Linden <ff.lin...@gmail.com> wrote:
> Hey guys,
>
> Thanks Nathan and Sebastian, I will compare the promoters. Just to clear it
> out, there is no way to know the amount of GFP inside the cell using only
> the spectrophotometer results? Lambert-beer equation doesn't apply in this
> case?

Beer-lambert does still apply, but measuring cell solution is going to
give a bit of a different result than measuring purified GFP. There
are going to be a lot of other molecules in the cytoplasm, in the
growth media, etc... the cell membrane will be in the way. So as long
as you compare your results to previously published (or your own
side-by-side) values that followed the same methodology (measuring
cell solution) you will get a meaningful answer. My guess is that even
something like cracking the cells open with a drop of soap or boiling
lysis is going to change the signal measured. Being consistent is key.

[Michael Tellier]

I did some comparison of GFP expression for comparing different constitutive promoters in E. coli in my previous lab. I used flow cytometry to rank the promoters but I came across this paper which uses other methods to rank constitutive promoters (http://www.pnas.org/content/102/36/12678.full   Tuning genetic control through promoter engineering). 

For an absolute quantification of the number of molecules, the only thing I remember was from a conference where people were using fluorescent microscopes to count the number of fluorescent molecules per cell for investigating promoter stochasticity. For flow cytometrry, I have been told that ~200 GFP molecules are needed for having a signal, below you cannot really differentiate from noise. My data with really weak promoters were in fact around the noise threshold.