Basu

Quantitative measure of HSL production by pArs LuxI construction ([http://parts.igem.org/Part:Bba_K1106008 Bba_K1106008])

Five different cultures were grown overnight:

• An E. coli culture carrying two plasmids that encode an incoherent feedforward system,

designed by Basu et al. (referencia Basu), which is inducible by Vibrio fischeri’s Lux system.

This plasmids were gently provided by Ron Weiss’ lab.

• A Rhizobium leguminosarum culture, grown at 30 ºC in TY medium, as a positive control.

• Two E. coli cultures (DH5alpha) harbouring the LuxI enzyme gene of Vibrio fischeri, under the

control of an arsenite inducible promoter (pArs). One of them was added with 1000 ppb of

arsenite.

Next day, E. coli culture carrying the incoherent feedforward plasmids was split in three tubes and

centrifuged. The cells were kept the cells, and the supernatant was discarded. Besides, the other

three cultures were centrifuged. The supernatant was kept and the cells were discarded. This

four conditioned media were used to resuspend the cells carrying Basu’s incoherent feedforward

plasmids.

Cells were incubated at 37 ºC and every 15 minutes we took an aliquot of each culture, until 105

minutes after the beginning of the induction.

Afterwards, we measured GFP production at each condition through time.