IGEM:UNAM/2009/Notebook/Modeling logbook Claudia/2010/03/26

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Objective: Ask for information about LovTap

I have been in touch with Dr. Devin Strickland and he kindly answered our doubts:

a.How important is the light intensity to activate LovTap?

You can estimate how effective your light source is by completely photoexciting and then quickly measuring the absorbance around 447 nm. See Fig. 2 of Salomon et al. (2000) for comparison.

b.Do you have any activation measure of LovTap in terms of photons concentration?

in our hands, 20 mW/cm^2 irradiance at 470 nm is just enough to fully activate LovTAP. (Coincidentally, this is about as much light as you can get out of the blue LEDs we used.) You should be able to calculate photon flux from this.

c.Besides using E. coli strain CY15058 as a trpR mutant, is there another important consideration about why did you choose it?

I used this strain because it is trpR deleted and has a trp reporter. The main disadvantage of this strain is that it has to be grown at 30 degC because of the reporter. If I remember correctly, you are using a different reporter, so any trpR deleted strain should be fine.

d.Is there a range spectrum to activate LovTap?

Generally anything from 400-500 nm is good for photoexcitation. You'll want to look at this reference for more details:

Salomon et al. Photochemical and mutational analysis of the FMN-binding domains of the plant blue light receptor, phototropin. Biochemistry (2000) vol. 39 (31) pp. 9401-10