"
Team:Berkeley/Applications
From 2013.igem.org
Over the summer, we looked into other possible interesting applications utilizing with our biological indigo dyeing process.
One application we envisioned was a fast and novel biosensor mechanism with our biological process utilizing our soluble indigo (indican)!
Current biosensors require transcriptional initiation on the reporter upon the detection of some substrate. 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 however dependent on transcription initiation on 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!
We will be utilizing our biological dyeing process to design our reporter system. In our reporter system, we will have our glucosidase enzyme that can be “turned off” or “on”, a build up store of indican in the cell, and receptors to detect the signal. Thus, when the signal is detected, our glucosidase enzyme will be “turned on,” and will subsequently convert the already present indican into indoxyl. Indoxyl will rapidly dimerize in the presence in the oxygen, allowing us to visually detect indigo as a result. This process is faster than the conventional mechanisms that require transcriptional initiation on the reporter upon the detection of some substrate for the following reasons:
1)Indican is already stored and present inside the cell
2)Glucosidase enzyme already present inside the cell
3)No need for additional translational and transcriptional steps to produce visible rep
An important aspect of this sensor mechanism is how we are controlling the enzyme from its off and on states. We devised a novel enzyme switch system 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.
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? The beta-glucosidase from B. Circulans that we are utilizing in our sensor process has never attempted to be split before.
Just how do we do split an enzyme?(Accession Number: 1QOX) To start, we first explored the structure of the glucosidase.
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.
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.
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.
