"
Team:Berkeley/Applications
From 2013.igem.org
| Line 19: | Line 19: | ||
<div id="1"><div class = "heading-large"><a name="Other Applications">Other Applications</a></div> | <div id="1"><div class = "heading-large"><a name="Other Applications">Other Applications</a></div> | ||
| - | <p> Over the summer, we looked into other possible interesting applications | + | <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 | + | One application we envisioned was a fast and novel biosensor mechanism that takes advantage of the enzymes which control indigo solubility, GT and GLU! |
<br><br> | <br><br> | ||
<div id="2"><div class = "heading"><a >Background</a></div> | <div id="2"><div class = "heading"><a >Background</a></div> | ||
| - | <p>Current biosensors | + | <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. |
While effective, this process can take many hours (in most cases 12+ hours) to see the reporter! In iGEM, several teams | 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 | 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 | 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 however dependent | + | of their substrate of interest ranging from fluorescent proteins to a variety of pigments. All of these projects are, however, dependent |
| - | on transcription initiation | + | 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> | indigo, in a matter of minutes!</p> | ||
</p> | </p> | ||
| Line 37: | Line 37: | ||
<div id="3"><div class = "heading"><a >Our Novel System</a></div> | <div id="3"><div class = "heading"><a >Our Novel System</a></div> | ||
| - | <p> | + | <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> | |
| - | + | ||
| - | in the oxygen | + | |
| - | that | + | |
<li><p>1)Indican is already stored and present inside the cell</p></li> | <li><p>1)Indican is already stored and present inside the cell</p></li> | ||
| - | <li><p>2) | + | <li><p>2)The glucosidase enzyme is already present inside the cell</p></li> |
| - | <li><p>3) | + | <li><p>3)There is no need for additional translational and transcriptional steps to produce visible output</p></li> |
<div style="text-align:center"> | <div style="text-align:center"> | ||
<img src="https://static.igem.org/mediawiki/2013/0/07/Enzymatic_Switch_Diagram.png" width="600" /> | <img src="https://static.igem.org/mediawiki/2013/0/07/Enzymatic_Switch_Diagram.png" width="600" /> | ||
</div> | </div> | ||
| - | <div id="4"><div class = "heading"><a >Novel Enzyme Switch through | + | <div id="4"><div class = "heading"><a >Novel Enzyme Switch through Phosphorylation and Binding Domains |
</a></div> | </a></div> | ||
<p>An important aspect of this sensor mechanism | <p>An important aspect of this sensor mechanism | ||
| - | is how we | + | is how we plan to shift the GLU enzyme between its off and |
| - | on states. | + | 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 |
| - | utilizing PKA phosphorylation. PKA (Protein Kinase A) recognizes a specific | + | |
| + | |||
| + | |||
| + | To go about doing this, we began to look at the consensus sequences of various sensor proteins, and attempted to find utilizing PKA phosphorylation. PKA (Protein Kinase A) recognizes a specific | ||
protein sequence (KR/KR/RN/pSPT/FILVY/I/F/D) to phosphorylate.</p> | protein sequence (KR/KR/RN/pSPT/FILVY/I/F/D) to phosphorylate.</p> | ||
Revision as of 02:53, 29 October 2013
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!
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!
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
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 To go about doing this, we began to look at the consensus sequences of various sensor proteins, and attempted to find utilizing PKA phosphorylation. 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.
So how will these different fragments be used to control the enzyme? To answer this question, we envisioned a split enzyme system, in which one split of the enzyme is carrying the binding domain, while the other split portion would be carrying the corresponding ligand. The requirement of this split enzyme is that the fragments must not be spontaneously assembled without the presence of its the correct ligand and binding domain.
