Figure 3. Using domain insertion to create a Fd switch.
(a) The ER-LBD was inserted after residue 35 in Ml-Fd to create sFd-35-ER. (b) Effect of 4-HT concentration on the complementation of E. coli EW11 by sFd-35-ER (k1/2 = 2.11 μM 4-HT), Ml-Fd, and Ml-Fd-C42A. Growth of sFd-35-ER is enhanced significantly above the C42A mutant in the presence of ≥6.25 μM 4-HT (p-values [6.25 μM-6×10–3, 12.5 μM-4×10–4, 25 μM-1×10–4, 50 μM-2×10–5] using a two-tailed, independent t-test). Symbols and error bars represent the mean and s.d., respectively (n=3 biologically independent samples). (c) The effect of 4-HT on the relative fluorescence of cells expressing sFd-35-ER-RFP. Upon addition of 4-HT in ethanol, there was no significant increase in fluorescence compared to ethanol alone (p-value > 0.01 using a two-tailed, independent t-test). Symbols and error bars represent the mean and s.d., respectively (n=3 biologically independent samples). All fluorescent reporters were amended to the C-terminus of proteins to maintain the context of the ribosomal binding sites and translation initiation43. (d) Effect of estrogen receptor modulators (50 μM) on complementation of E. coli EW11 by sFd-35-ER with 0 ng/mL aTc (white bars) or 200 ng/mL aTc (gray bars). The agonists (diethylstilbesterol, hexesterol, and 17β-estradiol) do not significantly enhance growth of EW11 cells compared to DMSO alone (p-value > 0.01 using a two-tailed, independent t-test), while the antagonists (4-hydroxytamoxifen, raloxifene, and lasoxifene) as well as bisphenol-A significantly enhance growth compared to DMSO alone. * represents significant growth (p-values [4-hydroxytamoxifen-1×10−4, raloxifene-5×10−6, lasoxifene-3×10−3, bisphenol-A-2×10−4] using a two-tailed, independent t-test). Bars and error bars represent the average and s.d., respectively (n=3 biologically independent samples).