Team:Berkeley/Applications

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             <li id="TitleID"> <a>Page: Indigo Biosynthesis</a> </li>
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             <li id="TitleID"> <a id="TitleID" href="https://2013.igem.org/Team:Berkeley/Applications">Other Applications</a> </li>
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            <li ><a href="#1">FMO Characterization</a></li>
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             <li ><a href="#2">Background</a></li>
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            <li ><a href="#2">Verification of Indigo</a></li>
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             <li ><a href="#3">Our Novel Sensing System</a></li>
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            <li ><a href="#3">Indigo Titer</a></li>
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             <li ><a href="#4">Novel Enzyme Switch through PKA Phosphorylation</a></li>
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             <li ><a href="#4">FMO Kinetics</a></li>
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             <li ><a href="#5">Indigo Toxicity</a></li>
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             <li ><a href="#6">References</a></li>
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  <div id="1"><div class = "heading-large"><a name="Other Applications">Other Applications</a></div>
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  <p> Over the summer, we looked into other possible interesting applications which would utilize parts of our biological indigo dyeing process. 
 +
  One application we envisioned was a fast and novel biosensor mechanism that takes advantage of the enzymes which control indigo solubility, GT and GLU!
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  <div id="1"><div class = "heading-large"><a name="Characterization of Indigo Biosynthesis">Characterization of Indigo Biosynthesis</a></div>
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<br><br>
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<div id="2"><div class = "heading"><a >Background</a></div>
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<p>Current biosensors operate by positively or negatively regulating the transcription levels of a given reporter gene. While there are many variations on this theme, they are all bounded by the time it takes for transcription and translation events to occur.
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  <div id="2"><div class = "heading"><a >Verification of Indigo</a></div>
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While effective, this process can take many hours (in most cases 12+ hours) to see the reporter! In iGEM, several teams
-
 
+
have used the biosensor approach in their iGEM projects developing biosensors ranging from cyanide and heavy metal sensors
 +
to rotting meat volatile sensors. These teams used a variety of colorimetric reporter elements as well to signify the detection
 +
of their substrate of interest ranging from fluorescent proteins to a variety of pigments. All of these projects are, however, dependent
 +
on transcription initiation of the reporter after the signal is detected. We have conceptually devised a novel mechanism to see our reporter compound,
 +
indigo, in a matter of minutes!</p>
 +
</p>
 +
 
 +
<br>
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<div id="3"><div class = "heading"><a >Our Novel Sensing System</a></div>
 +
 
 +
<p> In our hypothesized system, the e. coli would use FMO and GT to build up a store of indican in the cell. The cell would also contain a deactivated glucosidase enzyme that can be flipped into the "on” state in direct response to signal-detecting receptors, thus bypassing the lengthy transcription and translation events. When the signal is detected, our glucosidase enzyme will be turned on and shift the equilibrium from indican to indoxyl. As indoxyl builds up in the cell, it will begin to dimerize in the presence
 +
in the oxygen to create indigo, our colorometric output. This process is inherently faster than the conventional mechanisms
 +
that act via the cental dogma for the following reasons: </p>
 +
<li><p>1)Indican is already stored and present inside the cell</p></li>
 +
<li><p>2)The glucosidase enzyme is already present inside the cell</p></li>
 +
<li><p>3)There is no need for additional translational and transcriptional steps to produce visible output</p></li>
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<div style="text-align:center">
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<img src="https://static.igem.org/mediawiki/2013/0/07/Enzymatic_Switch_Diagram.png" width="600" />
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</div>
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 +
<div id="4"><div class = "heading"><a >Novel Enzyme Switch through Phosphorylation and Binding Domains
 +
</a></div>
 +
<p>An important aspect of this sensor mechanism
 +
is how we plan to shift the GLU enzyme between its off and
 +
on states. Our idea is to use phosphorylation to change the conformation of the enzyme from a catalytically inactive to a catalytically active state. To do this, we would have to find a way to make a sensor protein (like a serine-threonine kinase or a G-Protein Coupled Receptor) specifically phosphorylate the reporter protein whose state we want to regulate. Clearly though, just phosphorylating the reporter protein at a random location wouldn't necessarily regulate its activity. <br>
 +
We had two ideas to insure that the phosphorylation event would actually affect activity. The first would be to attach "arms" on both termini of the protein that would naturally bind one another and block the catalytic activity of the enzyme. Upon phosphorylation, the interactions between the arms would be disrupted and catalytic activity would be recovered.
 +
<br>
 +
The second idea, would make use of a split enzyme that can only come together when linked to peptides that have affinity for each other.
 +
To go about doing this, we began to look at PKA. PKA (Protein Kinase A) recognizes a specific
 +
protein sequence (KR/KR/RN/pSPT/FILVY/I/F/D) to phosphorylate.We exploited this fact to look for domains and ligands that match the phosphorylated sequence of PKA. Two different domains, BRCT and 14-3-3 contain corresponding binding ligands that match the PKA consensus sequence, and thus can be phosphorylated. Thus,  we designed these ligands, so that they will be recognized by PKA  and phosphorylated, and subsequently will bind to their respective binding domains. </p>
 +
<p>For controls, we designed a negative ligand that will not bind to
 +
the binding domain, and a positive ligand that is always turned on. </p>
 +
<div style="text-align:center">
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<img src="https://static.igem.org/mediawiki/2013/3/3e/Enzymatic_switch_berkeley.png" width="600" />
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</div>
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<!--<p>So how will these different fragments be used to control the enzyme?
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To answer this question, we envisioned a split enzyme system, in which
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one split of the enzyme is carrying the binding domain, while the
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other split portion would be carrying the corresponding ligand. The
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requirement of this split enzyme is that the fragments must not be
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spontaneously assembled without the presence of its the correct ligand
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and binding domain. </p>
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<div id="5"><div class = "heading"><a >Design and Implementation of Split Glucosidase
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</a></div>
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<p>We have seen that to control our enzyme from an off to on state, we will be
 +
utilizing split enzymes. But what if an enzyme has never been split before?
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The beta-glucosidase from B. Circulans that we are utilizing in our sensor process
 +
has never attempted to be split before.</p>
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<p>Just how do we do split an enzyme?(Accession Number: 1QOX) To start, we first explored the
 +
structure of the glucosidase.<p>
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<p>We identified loops as potential sites
 +
where the enzyme can be split, and can still maintain its functionality
 +
when put back together. 20 different cut sites were made, and
 +
subsequently synthesized in the lab. While these split constructs
 +
have been made and synthesized, they have yet not been tested
 +
experimentally.</p>
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<div id="6"><div class = "heading"><a >Alternative Method to Test Functionality of System
 +
</a></div>
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<p> We briefly tested our sensor mechanism with our binding domains and
 +
ligands with current existing split proteins. In current literature,
 +
versions of split beta-galactosidase and beta-lactamase are available
 +
in which the fragments are not spontaneous.</p>
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<p> To test this mechanism, constructs can be built with one split of the enzyme carrying the binding domain, with the
 +
other split portion carrying the corresponding ligand. </p>
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-->
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Latest revision as of 03:42, 29 October 2013

Other Applications

Over the summer, we looked into other possible interesting applications which would utilize parts of our biological indigo dyeing process. One application we envisioned was a fast and novel biosensor mechanism that takes advantage of the enzymes which control indigo solubility, GT and GLU!

Background

Current biosensors operate by positively or negatively regulating the transcription levels of a given reporter gene. While there are many variations on this theme, they are all bounded by the time it takes for transcription and translation events to occur. While effective, this process can take many hours (in most cases 12+ hours) to see the reporter! In iGEM, several teams have used the biosensor approach in their iGEM projects developing biosensors ranging from cyanide and heavy metal sensors to rotting meat volatile sensors. These teams used a variety of colorimetric reporter elements as well to signify the detection of their substrate of interest ranging from fluorescent proteins to a variety of pigments. All of these projects are, however, dependent on transcription initiation of the reporter after the signal is detected. We have conceptually devised a novel mechanism to see our reporter compound, indigo, in a matter of minutes!


Our Novel Sensing System

In our hypothesized system, the e. coli would use FMO and GT to build up a store of indican in the cell. The cell would also contain a deactivated glucosidase enzyme that can be flipped into the "on” state in direct response to signal-detecting receptors, thus bypassing the lengthy transcription and translation events. When the signal is detected, our glucosidase enzyme will be turned on and shift the equilibrium from indican to indoxyl. As indoxyl builds up in the cell, it will begin to dimerize in the presence in the oxygen to create indigo, our colorometric output. This process is inherently faster than the conventional mechanisms that act via the cental dogma for the following reasons:

  • 1)Indican is already stored and present inside the cell

  • 2)The glucosidase enzyme is already present inside the cell

  • 3)There is no need for additional translational and transcriptional steps to produce visible output

    • Novel Enzyme Switch through Phosphorylation and Binding Domains

    An important aspect of this sensor mechanism is how we plan to shift the GLU enzyme between its off and on states. Our idea is to use phosphorylation to change the conformation of the enzyme from a catalytically inactive to a catalytically active state. To do this, we would have to find a way to make a sensor protein (like a serine-threonine kinase or a G-Protein Coupled Receptor) specifically phosphorylate the reporter protein whose state we want to regulate. Clearly though, just phosphorylating the reporter protein at a random location wouldn't necessarily regulate its activity.
    We had two ideas to insure that the phosphorylation event would actually affect activity. The first would be to attach "arms" on both termini of the protein that would naturally bind one another and block the catalytic activity of the enzyme. Upon phosphorylation, the interactions between the arms would be disrupted and catalytic activity would be recovered.
    The second idea, would make use of a split enzyme that can only come together when linked to peptides that have affinity for each other. To go about doing this, we began to look at PKA. PKA (Protein Kinase A) recognizes a specific protein sequence (KR/KR/RN/pSPT/FILVY/I/F/D) to phosphorylate.We exploited this fact to look for domains and ligands that match the phosphorylated sequence of PKA. Two different domains, BRCT and 14-3-3 contain corresponding binding ligands that match the PKA consensus sequence, and thus can be phosphorylated. Thus, we designed these ligands, so that they will be recognized by PKA and phosphorylated, and subsequently will bind to their respective binding domains.

    For controls, we designed a negative ligand that will not bind to the binding domain, and a positive ligand that is always turned on.

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