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Team:Imperial College London/Reporters
From 2011.igem.org
Reporters
Fluorescent reporters are an important tool in molecular biology, as they are frequently used to label various intracellular processes. In synthetic biology, fluorescent reporters are often used as the output of a genetic circuit, for example to signal the detection of a chemical.
As part of our iGEM project, we implemented a new fluorescent reporter, Dendra 2. In addition, we introduced a new coding sequence for superfolder GFP that is codon optimised for E.coli.
To further characterise these parts, we have conducted a thermostability assay to determine the temperature at which these proteins denature and cease to fluoresce. This was done by first expressing the fluorescent proteins in E.coli and then growing them in large batch cultures. The cultures were centrifuged, and then the resulting pellet was resuspended in Tris lysis buffer. A sonicator was used to lyse the cells, and then the resulting mixture was centrifuged again to remove cell debris. The supernatants containing the fluorescent proteins were then kept at 4°C to preserve them, since freezing would result in protein degradation.
The protein solutions were then heated in a PCR Thermocycler along a temperature gradient. The samples were analysed by fluorescence spectroscopy, and then the relative fluorescence was plotted against temperature to find the temperature at which the proteins denature
Dendra 2
Dendra 2 is a green fluorescent protein that can be irreversibly single-photon photoconverted into a red fluorescent protein.
Photoconversion
Thermostability
Superfolder GFP
This is a variant of GFP that has been engineered to be faster folding so that it can be used for tagging proteins more efficiently. The variant that we're submitting to the registry has been codon optimised for E.coli.
