Light-regulated gene expression systems allow controlling gene expression in space and time with high accuracy.Contrary to previous synthetic light sensors that incorporate two-component systems which require localiza...Light-regulated gene expression systems allow controlling gene expression in space and time with high accuracy.Contrary to previous synthetic light sensors that incorporate two-component systems which require localization at the plasma membrane,soluble one-component repression systems provide several advantageous characteristics.Firstly,they are soluble and able to diffuse across the cytoplasm.Secondly,they are smaller and of lower complexity,enabling less taxing expression and optimization of fewer parts.Thirdly,repression through steric hindrance is a widespread regulation mechanism that does not require specific interaction with host factors,potentially enabling implementation in different organisms.Herein,we present the design of the synthetic promoter P_(EL)that in combination with the light-regulated dimer EL222 constitutes a onecomponent repression system.Inspired by previously engineered synthetic promoters and the Escherichia coli lacZYA promoter,we designed P_(EL)with two EL222 operators positioned to hinder RNA polymerase binding when EL222 is bound.P_(EL)is repressed by EL222 under conditions of white light with a light-regulated repression ratio of five.Further,alternating conditions of darkness and light in cycles as short as one hour showed that repression is reversible.The design of the P_(EL)EL222 system herein presented could aid the design and implementation of analogous one-component optogenetic repression systems.Finally,we compare the P_(EL)-EL222 system with similar systems and suggest general improvements that could optimize and extend the functionality of EL222-based as well as other one-component repression systems.展开更多
基金the fluorescence spectrum of EL222,and Adam Wegelius is gratefully acknowledged for experimental assistance.AJ acknowledges funding from FP7-ICT-610730(EVOPROG)FP7-KBBE-613745(PROMYS)+2 种基金H2020 Marie Sklodowska-Curie Actions 642738(MetaRNA)Biotechnology and Biological Biological Sciences Research Council(BBSRC)BB/P020615/1(EVO-ENGINE),Engineering and Physical Sciences Research Council-Biotechnology and Biological Sciences Research Council(EPSRC-BBSRC)BB/M017982/1(WISB centre),and the departmental allocation from the School of Life Sciences(U.Warwick)PL acknowledges funding from the Knut och Alice Wallenbergs Stiftelse(project MoSE,No.2011.0067)and the Swedish Energy Agency(No.11674-5).
文摘Light-regulated gene expression systems allow controlling gene expression in space and time with high accuracy.Contrary to previous synthetic light sensors that incorporate two-component systems which require localization at the plasma membrane,soluble one-component repression systems provide several advantageous characteristics.Firstly,they are soluble and able to diffuse across the cytoplasm.Secondly,they are smaller and of lower complexity,enabling less taxing expression and optimization of fewer parts.Thirdly,repression through steric hindrance is a widespread regulation mechanism that does not require specific interaction with host factors,potentially enabling implementation in different organisms.Herein,we present the design of the synthetic promoter P_(EL)that in combination with the light-regulated dimer EL222 constitutes a onecomponent repression system.Inspired by previously engineered synthetic promoters and the Escherichia coli lacZYA promoter,we designed P_(EL)with two EL222 operators positioned to hinder RNA polymerase binding when EL222 is bound.P_(EL)is repressed by EL222 under conditions of white light with a light-regulated repression ratio of five.Further,alternating conditions of darkness and light in cycles as short as one hour showed that repression is reversible.The design of the P_(EL)EL222 system herein presented could aid the design and implementation of analogous one-component optogenetic repression systems.Finally,we compare the P_(EL)-EL222 system with similar systems and suggest general improvements that could optimize and extend the functionality of EL222-based as well as other one-component repression systems.
