FIG 5.

Dichromatic optogenetic module for regulating intracellular c-di-GMP levels comprising a far-red/near-infrared-light-regulated DGC, BphS, and a blue-light-activated PDE, EB1. (A) Structure of the synthetic bphS-bphO-eb1 operon. A semicircle in front of each gene indicates a RBS; the T sign at the end of the operon indicates a transcription terminator. (B) c-di-GMP-dependent curli fimbria synthesis in E. coli BL21(DE3). Fresh colonies of BL21(DE3) transformants carrying the indicated plasmids were grown on Congo red plates for 4 days at 30°C. 1, pMslr1143 (constitutive DGC; positive control); 2, pbSHE (bphS-bphO-eb1); 3, pbSHY2 (bphS-bphO-yhjH; RBS2); 4, pbSHY3 (bphS-bphO-yhjH; RBS3); 5, pbSHY4 (bphS-bphO-yhjH; RBS4); 6, pET23a (empty vector) (negative control). Dark indicates growth in the dark (plate covered in aluminum foil), red indicates colonies irradiated with pulsed (30 s of light and 120 s of dark) red (650-nm) light, and blue indicates colonies irradiated with pulsed (10 s of light and 60 s of dark) blue (465-nm) light.