Difference between revisions of "Team:Stanford-Brown/SB16 BioSensor FQsensor"
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| − | <div class="figure-legend"> | + | <div class="figure-legend">Figure 1: Aptamer-Based Fluorophore-Quencher Small Molecule Biosensing Probe. Two modified oligonucleotides are the core of this system: 1. one biotinylated DNA sequence contains the aptamer-folding domain (the red sequence) for a target sensing molecule (e.g. ATP, Lead, Perchlorate) along with a fluorophore (F) for signaling, 2. a second strand contains a quencher (Q) to suppress the fluorescent probe when in proximity. This system is attached to a surface by the streptavidin-biotin (S-B) complex. Initially at standard conditions, these two strands hybridize to quench the fluorophore; however, in the presence of the target small molecule, the thermodynamically favored state is the folded DNA aptamer, which sterically opens the fluorophore.</div> |
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Revision as of 20:17, 7 October 2016
Introduction
The Fluorophore-Quencher system is a variation on the same idea that the IRES system was predicated on: use an aptamer as a sensing domain, and something else as an expression platform to signal that the aptamer had bound its target. In the case of the fluorophore-quencher, we synthesized a fluorescent molecule (fluorophore) directly onto the 5’ end of the aptamer, and a biotinylated the 3’ end. From there, we ordered an oligo that was complementary to the 5’ end of the aptamer and had a quencher synthesized onto the end of it. When both oligos were allowed to incubate in a streptavidin plate, the result was the complex in Figure 1 part A, where the sensor is attached to a solid surface, and its fluorescence is quenched. When target ligand is introduced, the aptamer undergoes conformational change as it folds around the ligand. This conformational change sterically displaces the quencher oligo and exposes the fluorophore, thus emitting detectable signal.
Figure 1: Aptamer-Based Fluorophore-Quencher Small Molecule Biosensing Probe. Two modified oligonucleotides are the core of this system: 1. one biotinylated DNA sequence contains the aptamer-folding domain (the red sequence) for a target sensing molecule (e.g. ATP, Lead, Perchlorate) along with a fluorophore (F) for signaling, 2. a second strand contains a quencher (Q) to suppress the fluorescent probe when in proximity. This system is attached to a surface by the streptavidin-biotin (S-B) complex. Initially at standard conditions, these two strands hybridize to quench the fluorophore; however, in the presence of the target small molecule, the thermodynamically favored state is the folded DNA aptamer, which sterically opens the fluorophore.
Data
Sample
We conclude from this data that this sensor worked well with high ATP concentrations, but with further research could almost certainly be optimized to work better at lower concentrations. Advantages of this system include its customizability (theoretically it should work to detect any molecule for which there exists an aptamer) and its ability to retain sensory functionality while bound to a solid surface. Disadvantages include only detecting high concentrations of target ligand (at least currently, though this could theoretically be at least somewhat mitigated with further efforts at system optimization), and the fact that this system is fundamentally synthetic, limiting its ability to be easily and cheaply replicated in vivo like other bio-bricked parts. Instead, expensive new constructs have to be ordered to build each new sensor.
