By responding to a wide range of small molecules, riboswitches are capable of regulating gene expression at the translational level. This property has led to the widespread exploration of their usages as gene regulation device in synthetic biology.
The application of riboswitches is currently hindered by several disadvantages, including context-dependent performance and limited dynamic range. When employ a particular riboswitch on various ORFs which are different with its natural one, the structure of the aptamer domain may be disrupted, resulting in loss of ligand response.
In this way, the riboswitch is still not considered as a plug-and-play modular component
What did we do?
To make riboswitch more accessible to future synthetic biologists and iGEM teams, our project focused on a standardized principle to design modular riboswitch.
The certain sequences were found and chosen to stabilize the structure of the riboswitch, which named ‘Stabilizer’. When constructing this fragment at the downstream of the riboswitch part as an integrated context, it could work as a kind of ‘insulator’ to protect the functional structure of the riboswitch. Using docking matrix, the appropriate length is determined.
By optimizing an existing artificial mechanism known as the ‘Ribo-attenuator’ which could avoid the unpredictable folding abnormality of the desired protein caused by the N-terminal fused peptide translated from the ‘Stabilizer’. We developed a complete set of design principles and models for the rational design of the attenuators. More ‘Tuners’ are designed by our Ribo-innovative rational design method to make diverse dynamic ranges and the maximum expression levels, which provides an ideal modular riboswitch toolkit for multifunctional genetic regulation.
Due to the general difficulty of degradation of small molecules (inducer), it is not easy turn down the function of riboswitch without the medium replacement which is impractical in many industrial applications. Here, we set up a model to design different asRNAs which could target different regions of the riboswitch to control its function. We finally achieved a feasible design to regulate the on-off state of the riboswitch, which demonstrates the prediction capability of our modeling.
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