Engineer Biomolecular Switches That Respond To External Signals

Synthetic biology has become an important discipline over the past few years.It is a hybrid field that could combine various functional components in the genetic circuits to regulate gene networks.Synthetic biology has several significant characteristics that make it become more attractive among various disciplines in life science,it could create unusual genetic circuits by combining the optimal features of natural systems with designs and development,thus make the genetic circuits show some remarkable properties such as extensible,comprehensible and helpful to fulfill specific function.Furthermore,a variety of synthetic biology components have been engineered successfully to identify signals and control gene expression specifically.Herein,we use several strategies to construct module components to regulate gene expression,the elements in our experiments could identify the external stimuli signals and cause specific response in vivo,moreover,the regulators we engineered show robust and controllable property.The first strategy we utilized is to engineer artificial RNA switches that response to small chemical molecules.In the study,we adopt an in vitro evolution strategy that could be able to combine the aptamer domain and the expression platform thus to construct a riboswitch,the riboswitch we created has the ability to distinguish itself from others based on the potential of high affinity to the specific ligand,and show the capacity to confer control over gene expression.During the past years,many DNA and RNA-based aptamers obtained based on SELEX and in vitro selection show high affinity to ligands.However,it's still difficult to select for conformational change upon ligand binding directly,thus engineering riboswitches de novo that respond to chemical compounds has remained challenge.In the study,we construct an in vitro screening and selection strategy based on SELEX technique to select RNA switches with switchable property directly.The library we engineered based on the previously reported theophylline aptamer.The specific sequences selected from in vitro selection completely could show significant conformational change and the interaction of RNA-DNA is robust in the presence of theophylline.Furthermore,the selected RNA switches could have the characteristics of riboswitch activity similar to that of natural riboswitches.The strategy we engineered based on SELEX and in vitro selection could helpful for designing RNA switches with more complex and external signals responsive conformational change.Further,photo-activation of cellular proteins has become a powerful strategy for studying complex biological systems based on precise spatial and temporal coordination recently.In the study,we engineer light-activatable genetic switches based on the bacterium phage T7 RNA polymerase(T7 RNAP),in which the activity of T7 RNAP is controlled by optical signal directly.The photoactivatable genetic switches are constructed with a design strategy of splitting the polymerase into two fragments and fusing with the regulatory domains to regulate the process of reconstitutions.The genetic switches show the robust switchable characteristics with excellent light-on/dark-off properties,when the regulatory domains are the light-activatable VVD domain and its variants.Furthermore,for the best split position we found,the light-induced genetic switches could exploit either the light-activatable interactions between the VVD domains or allosteric effects to control the polymerase activity.Besides,the split position shows high modularity,the fragments split from the specific split position could be combined with different regulatory domains such as those with chemically inducible interaction,thus enabling construct chemically controlled switches.To summarize,the engineered genetic switches based on T7 RNA polymerase are powerful tools,it's helpful for implementing light-activated gene expression in different contexts.Moreover,understanding of the split positions and domain organizations may facilitate future engineering studies on this and on related proteins.In addition,one of the central goals of synthetic biology is to implement diverse cellular functions by regulating gene expression predictably.Until now,although various studies mainly focus on the regulators engineered based on protein,recent advances in the study of RNA conformational change and function have make the use of RNA-based regulators become more convenient and efficient.In contrast to the protein-based regulators,RNA-based regulators due to easier to design and construct,thus provide an advantage and make the RNA-based regulators have lower burden to the host cells potentially and show significant orthogonality.Herein,we combine the CRISPR-dCas9 system and the de novo engineered antisense RNAs(asRNAs)in E.coli strains to repress or de-repress gene expression in a programmable manner.We found that the gene expression repressed by CRISPR-dCas9 system could be de-pressed by asRNAs,the asRNAs we designed could interact with the single guide RNA(sgRNA)specifically.Moreover,we demonstrate that the de-repression levels could be tunable by engineering asRNA that make the asRNA interact with different regions of the sgRNA.The distinctive regulation strategy taking advantage of CRIPSR-dCas9 system and asRNA system in the genetic circuits,could be utilized to repress or de-repress various gene expression reversibly and simultaneously,and it's helpful for rational and tunable regulation of cellular functions.