Difference between revisions of "Team:BostonU/Temporal Control/Current Methods"

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<h3> Current Methods</h3>
 
<h3> Current Methods</h3>
  
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<p>One way to temporally control a protein is to physically split it and fuse halves of the proteins to domains that naturally dimerize in the presence of an inducer; and absence or presence of inducer will modulate when the protein is inactive or active, respectively. </p>
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<p>Cells naturally regulate protein expression at various stages of protein expression. Generally, protein activity control in synthetic systems arises as two particular time points: pre-transcription and post-translation. Pre-transcriptional control involves controlling protein activity through protein expression. If a protein is not even transcribed, it is not expressed and thus not active; in contrast, if it is expressed, it has full natural activity. Post-translational control involves controlling activity of a protein after it has been translated - this often uses chemical or genetic modifications that control activity after expression. </p>
  
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<p>Pre-translational control can involve controlling protein expression through an inducible promoter, which typically involves a transcription factor interacting with a small molecule to either repress or activate downstream gene expression. For the protein to be expressed, it has to be transcribed and translated. Post-translational control methods can technically yield a faster response to induction by bypassing the process of transcribing and translating the sequence encoding the protein. </p>   
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<td>Another way to control proteins is to put it under the control of an inducible promoter.  This is a type of transcriptional control. Transcriptional control is an effective method of creating single logic circuits in cells.  </td>
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<td><p>INSERT SIMPLE AND/OR/NOR GATE WITH INDUCIBLE PROMOTER LOGIC GATE</p></td>
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<td><p>SHOW IMAGE OF HIGHER ORDER INDUCIBLE PROMOTER LOGIC GATE</p></td>
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<td>However, building nested circuits and higher order logic becomes challenging with this system.</td>
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<p>The nested logic causes delays in response with each layer of logic. In post-translational modulation systems, the proteins are fully functional as a response to the proper inputs.</p>   
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<p>For the split integrase + RDF and saCas9 systems that we explored, it was important that for the input control to be completely orthogonal to the cellular system. The integrase + RDF system would not supply any output with inducible promoters that allowed transcription before an input signal was received.  In other words, any leakiness at all in the inducible promoter system with integrases and RDFs would cause transcription and translation and turn the gene immediately off. Post-translational control by splitting gives us tighter control over the integrase and RDF system. </p>
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<p>For proteins that manipulate the genome, basal activity can be a big concern. If there was any “accidental” expression a few of these proteins (for example through leaky expression under an inducible promoter), the genetic state can easily be switched since the expressed protein is naturally active. However, using post-translational control, the protein can be expressed in an inactive form and only activated in presence of the inducer. This method also has practical limitations, but optimization may potentially lead to lower basal activity.</p>
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<p>While there are advantages and disadvantages to both of these methods of protein activity control, they are not mutually exclusive. Combining these systems can lead to more intricate genetic manipulation and genome engineering applications. This summer, our team decided to study post-translational control of genetically-modifying proteins and how these might be useful tools for different synthetic biology applications.</p>
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Revision as of 01:11, 18 September 2015

Overview Current Methods Our Workflow

Current Methods

Cells naturally regulate protein expression at various stages of protein expression. Generally, protein activity control in synthetic systems arises as two particular time points: pre-transcription and post-translation. Pre-transcriptional control involves controlling protein activity through protein expression. If a protein is not even transcribed, it is not expressed and thus not active; in contrast, if it is expressed, it has full natural activity. Post-translational control involves controlling activity of a protein after it has been translated - this often uses chemical or genetic modifications that control activity after expression.

Pre-translational control can involve controlling protein expression through an inducible promoter, which typically involves a transcription factor interacting with a small molecule to either repress or activate downstream gene expression. For the protein to be expressed, it has to be transcribed and translated. Post-translational control methods can technically yield a faster response to induction by bypassing the process of transcribing and translating the sequence encoding the protein.

For proteins that manipulate the genome, basal activity can be a big concern. If there was any “accidental” expression a few of these proteins (for example through leaky expression under an inducible promoter), the genetic state can easily be switched since the expressed protein is naturally active. However, using post-translational control, the protein can be expressed in an inactive form and only activated in presence of the inducer. This method also has practical limitations, but optimization may potentially lead to lower basal activity.

While there are advantages and disadvantages to both of these methods of protein activity control, they are not mutually exclusive. Combining these systems can lead to more intricate genetic manipulation and genome engineering applications. This summer, our team decided to study post-translational control of genetically-modifying proteins and how these might be useful tools for different synthetic biology applications.