Team:Peking R/Project/RNAToolkit

From 2011.igem.org

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   <p class="mainbody">In recent years, RNA devices have emerged  as powerful tools to regulate gene expression <em>in vivo</em>, and particularly, ligand-responsive riboswitches/ribozymes  enable us to manipulate translation strength of specific genes upon different  concentrations of ligands. Ligand-responsive riboswitches/ribozymes regulate  the translation rate of downstream gene by changing conformations, cleaving or  splicing upon external addition of ligand. Compared with transcriptional and  post-translational regulation, riboswitches/ribozymes function through  allostery of RNA structure, which requires little or no assistance from  proteins, so the regulation mechanism is relatively simpler and their functions  are more decoupled from native biological activities.</p>
   <p class="mainbody">In recent years, RNA devices have emerged  as powerful tools to regulate gene expression <em>in vivo</em>, and particularly, ligand-responsive riboswitches/ribozymes  enable us to manipulate translation strength of specific genes upon different  concentrations of ligands. Ligand-responsive riboswitches/ribozymes regulate  the translation rate of downstream gene by changing conformations, cleaving or  splicing upon external addition of ligand. Compared with transcriptional and  post-translational regulation, riboswitches/ribozymes function through  allostery of RNA structure, which requires little or no assistance from  proteins, so the regulation mechanism is relatively simpler and their functions  are more decoupled from native biological activities.</p>
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   <p class="mainbody">In order to fulfill the goal of establishing  an extensible and versatile platform for softcoding of genetic program, our  team reached out to a great extent to search for available ligand-responsive  riboswitches/ribozymes that meet our criteria and selected them into our RNA  toolkit. </p>
+
   <p class="mainbody">In order to fulfill the goal of establishing  an extensible and versatile platform for softcoding of genetic program, our  team reached out to a great extent to search for available ligand-responsive  riboswitches/ribozymes that meet our criteria and selected them into our genetic rheostat. </p>
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   <p class="mainbody">RNA controller candidates must meet two basic criteria: firstly, they  should possess a relatively plain dose-response curve, which would allow for  precise translation strength modulation within a wide range of ligand  concentration; secondly, ligands they recognize should be genetically and  biochemically orthogonal to the host cells, in our case, <em>E.coli</em> cells, as much as possible. </p>
+
   <p class="mainbody">Genetic rheostat candidates must meet two basic criteria: firstly, they  should possess a relatively plain dose-response curve, which would allow for  precise translation strength modulation within a wide range of ligand  concentration; secondly, ligands they recognize should be genetically and  biochemically orthogonal to the host cells, in our case, <em>E.coli</em> cells, as much as possible. </p>
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   <p class="mainbody">Two candidates emerged as promising RNA  tools that satisfied our requirements: thiamine pyrophosphate (TPP)-responsive  hammerhead ribozymes and theophylline-responsive riboswitches. By altering the  upstream promoter and downstream coding sequence of the RNA controllers, we  demonstrated that their performance was independent from sequence context,  which proved the modularity of these RNA devices.</p>
+
   <p class="mainbody">Two candidates emerged as promising genetic rheostates that satisfied our requirements: thiamine pyrophosphate (TPP)-responsive  hammerhead ribozymes and theophylline-responsive riboswitches. By altering the  upstream promoter and downstream coding sequence of the genetic rheostates, we  demonstrated that their performance was independent from sequence context,  which proved the modularity of these RNA devices.</p>
-
   <p class="mainbody">To further extend the range of application  of our RNA toolkit, we created a ribozyme that functions with a different  mechanism, which has an extreme low level of backgrounds. We substituted the  aptamer domain of c-di-GMP group Iintron to theophylline-responsive aptamer,  thus invented a group I intron that senses theophylline to perform splicing  function. </p>
+
   <p class="mainbody">To further extend the range of application  of our genetic rheostat, we created a ribozyme that functions with a different  mechanism, which has an extreme low level of backgrounds. We substituted the  aptamer domain of c-di-GMP group Iintron to theophylline-responsive aptamer,  thus invented a group I intron that senses theophylline to perform splicing  function. </p>
   <p class="mainbody">Moreover, we introduced a general method to  evolve hammerhead ribozyme that senses a new ligand. By coupling an adenine  aptamer with hammerhead ribozyme and randomizing nucleotides in the linker  domian, we evolved new hammerhead ribozymes through dual selection, whose  self-cleavage could be regulated by adenine. </p>
   <p class="mainbody">Moreover, we introduced a general method to  evolve hammerhead ribozyme that senses a new ligand. By coupling an adenine  aptamer with hammerhead ribozyme and randomizing nucleotides in the linker  domian, we evolved new hammerhead ribozymes through dual selection, whose  self-cleavage could be regulated by adenine. </p>
   <p class="mainbody">In summary, our project provided new design  principles for rational or semi-rational design of riboswitches/ribozymes. </p>
   <p class="mainbody">In summary, our project provided new design  principles for rational or semi-rational design of riboswitches/ribozymes. </p>

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Brief introduction to RNA toolkit


 

 

 

 

 

click the RNA Controllers' name to learn more


In recent years, RNA devices have emerged as powerful tools to regulate gene expression in vivo, and particularly, ligand-responsive riboswitches/ribozymes enable us to manipulate translation strength of specific genes upon different concentrations of ligands. Ligand-responsive riboswitches/ribozymes regulate the translation rate of downstream gene by changing conformations, cleaving or splicing upon external addition of ligand. Compared with transcriptional and post-translational regulation, riboswitches/ribozymes function through allostery of RNA structure, which requires little or no assistance from proteins, so the regulation mechanism is relatively simpler and their functions are more decoupled from native biological activities.

In order to fulfill the goal of establishing an extensible and versatile platform for softcoding of genetic program, our team reached out to a great extent to search for available ligand-responsive riboswitches/ribozymes that meet our criteria and selected them into our genetic rheostat.

Genetic rheostat candidates must meet two basic criteria: firstly, they should possess a relatively plain dose-response curve, which would allow for precise translation strength modulation within a wide range of ligand concentration; secondly, ligands they recognize should be genetically and biochemically orthogonal to the host cells, in our case, E.coli cells, as much as possible.

Two candidates emerged as promising genetic rheostates that satisfied our requirements: thiamine pyrophosphate (TPP)-responsive hammerhead ribozymes and theophylline-responsive riboswitches. By altering the upstream promoter and downstream coding sequence of the genetic rheostates, we demonstrated that their performance was independent from sequence context, which proved the modularity of these RNA devices.

To further extend the range of application of our genetic rheostat, we created a ribozyme that functions with a different mechanism, which has an extreme low level of backgrounds. We substituted the aptamer domain of c-di-GMP group Iintron to theophylline-responsive aptamer, thus invented a group I intron that senses theophylline to perform splicing function.

Moreover, we introduced a general method to evolve hammerhead ribozyme that senses a new ligand. By coupling an adenine aptamer with hammerhead ribozyme and randomizing nucleotides in the linker domian, we evolved new hammerhead ribozymes through dual selection, whose self-cleavage could be regulated by adenine.

In summary, our project provided new design principles for rational or semi-rational design of riboswitches/ribozymes.


Existing Natural RNA Controllers
  TPP-regulated hammerhead ribozyme
  THEOPHYLLINE-responsive riboswitch

Engineered RNA controllers

  RIBOZYME:engineered group I intron with a theophylline hammerhead ribozyme
  SELECTION:adenine hammerhead ribozyme obtained from screening