br Experimental design materials and methods br Conflicts of

Experimental design, materials and methods
Conflicts of interest
Acknowledgments This study was founded by grants from CNPq (Conselho Nacional do Desenvolvimento Científico e Tecnológico - process 302885/2011-0) and CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior). Mirian Salvador metalloproteinase the recipient of a CNPq Research Fellowship. Caroline Calloni and Luciana Fernandes Silva Santos are the recipients of CAPES Research Fellowships. Luana Soares Martínez is the recipient of FAPERGS Research Fellowship.
Data To properly appreciate the high thermal sensitivity of TRPA1s from infrared-sensing snakes such as rattlesnakes and boas [1] in comparison with Drosophila melanogaster TRPA1 [2,3], a new analysis for temperature coefficient Q10 (fold increase of current upon 10°C temperature shift) was recently devised and called “Q10 scanning” [1]. By reducing the noise level of the temperature-evoked current through Gaussian filtering and shortening the interval between the two temperatures of Q10 calculation, the Q10 scanning method was tuned for higher Q10 sensitivity and more precise estimation of temperature parameters as presented here (Fig. 1). As alternative ways of analyzing temperature sensitivity of TRPA1s, the thermally induced current traces were fitted to equations to acquire the Boltzmann slope factor (Fig. 2) and the molar heat capacity change (Fig. 3).
Experimental design, materials and methods The temperature-evoked current traces were previously acquired [1] by conducting two-electrode voltage clamping [4,5] on Xenopus laevis oocytes expressing each TRPA1. The current traces were data-reduced by the factor of 10 through replacing 10 data points with their average value, filtered by the Gaussian low pass filter at 1Hz, and further smoothened by averaging neighboring 50 data points in order to minimize the noise level. For each t1 temperature, Q10 was obtained by its definition (Q10=(I2/I1)) with the use of two current and temperature data points apart from each other by 20,100, and 200 data points corresponding to 0.1, 0.5, and 1s or 0.05, 0.25, and 0.5°C, respectively. The calculated Q10s were plotted as function of temperature t1. The peak Q10 was computed by averaging 100 data points flanking the maximum Q10. The temperature where Q10 starts to increase or which produces the maximum Q10 was referred to as deflection temperature (Td) or peak temperature (Tp), respectively. Td and Tp were compared with the Arrhenius activation threshold temperature (Tth) [6]. To obtain the Boltzmann slope factors, traces were fitted to the Boltzmann equation using Sigmaplot12.0. To acquire the change in molar heat capacity Δ, the temperature-evoked currents were transformed to traces of lnK by plotting lnI/(Imax−I) as function of temperature, assuming for simple comparative analyses that Imax (peak current amplitude) is obtained with open probability of 1. The steepest part of the lnK graph was fitted with Sigmaplot 12.0 to the equation of “lnK=ΔS°0/R–Δ[1−T0/T+ln(T0/T)]/R” as function of T (temperature in Kelvin) to inquire the change of the molar heat capacity [7].
Acknowledgments I would like to thank Dr. Hana Cho for insightful comments to this manuscript and introduction to the data analyses used in the paper. This work was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2015R1D1A1A01057288) and a research fund from Samsung Biomedical Research Institute (SMX1150561) to KJK.
Data A cyclohexanone-responsive expression platform was designed based on elements of the cyclohexanol biodegradation pathway of Acinetobacter johnsonii (Fig. 1A). The segments bearing the complete chnR and P promoter DNA of A. johnsonii NCIMB 9871 were edited in silico to obtain a standardized SEVA (Standard European Vector Architecture [2]) expression cargo (Fig. 1B), and assembled to yield plasmid pSEVA2311 (Fig. 2 and Table 1). Expression data were generated to validate this plasmid. The gene encoding the monomeric and superfolder green fluorescent protein (msf•GFP) was inserted in vector pSEVA2311 (Fig. 3A), and the transcriptional activation of the ChnR/P expression system upon addition of cyclohexanone was evaluated in a wild-type Escherichia coli strain (Fig. 3B and C). Key features of this expression vector include [i] a very low expression level in the absence of inducer, [ii] high transcriptional capacity, [iii] an induction kinetics very similar in both minimal and rich culture media, and [iv] linear accumulation of the reporter product along time.