Red Light Responsive\nCre Recombinase for Bacterial\nOptogenetics

Optogenetic tools have been used in a wide range of microbial\nengineering\napplications that benefit from the tunable, spatiotemporal control\nthat light affords. However, the majority of current optogenetic constructs\nfor bacteria respond to blue light, limiting the potential for multichromatic\ncontrol. In addition, other wavelengths offer potential benefits over\nblue light, including improved penetration of dense cultures and reduced\npotential for toxicity. In this study, we introduce OptoCre-REDMAP,\na red light inducible Cre recombinase system in Escherichia\ncoli. This system harnesses the plant photoreceptors\nPhyA and FHY1 and a split version of Cre recombinase to achieve precise\ncontrol over gene expression and DNA excision. We optimized the design\nby modifying the start codon of Cre and characterized the impact of\ndifferent levels of induction to find conditions that produced minimal\nbasal expression in the dark and induced full activation within 4\nh of red light exposure. We characterized the system’s sensitivity\nto ambient light, red light intensity, and exposure time, finding\nOptoCre-REDMAP to be reliable and flexible across a range of conditions.\nIn coculture experiments with OptoCre-REDMAP and the blue light responsive\nOptoCre-VVD, we found that the systems responded orthogonally to red\nand blue light inputs. Direct comparisons between red and blue light\ninduction with OptoCre-REDMAP and OptoCre-VVD demonstrated the superior\npenetration properties of red light. OptoCre-REDMAP’s robust\nand selective response to red light makes it suitable for advanced\nsynthetic biology applications, particularly those requiring precise\nmultichromatic control.