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Team:Imperial College London/Reporters
From 2011.igem.org
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<iframe width="420" height="315" src="http://www.youtube.com/embed/EFYe296tu1I?rel=0" frameborder="0" allowfullscreen></iframe> | <iframe width="420" height="315" src="http://www.youtube.com/embed/EFYe296tu1I?rel=0" frameborder="0" allowfullscreen></iframe> | ||
<p><i>Video 2:A video of another photoconversion of Dendra in E. coli cells that have been taken up into Arabidopsis roots can be seen.</i></p> | <p><i>Video 2:A video of another photoconversion of Dendra in E. coli cells that have been taken up into Arabidopsis roots can be seen.</i></p> | ||
| + | <p><b>How did we photoconvert Dendra 2 whilst exciting and detecting RFP?</b></p> | ||
| + | <p>Using a FluoroMax-3 machine, and enlisting the expertise of Tom Wilson, we used Tom's 405nm laser to photoconvert Dendra 2. This was done by aiming the laser into the sample cuvette | ||
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<h4>Thermostability</h4> | <h4>Thermostability</h4> | ||
<h3>Superfolder GFP</h3><p><img align="right" class="border" src="https://static.igem.org/mediawiki/2011/6/60/ICL_sfGFP_PURE.png" width="100px" height="300px" /></p> | <h3>Superfolder GFP</h3><p><img align="right" class="border" src="https://static.igem.org/mediawiki/2011/6/60/ICL_sfGFP_PURE.png" width="100px" height="300px" /></p> | ||
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<p>mRFP is a monomeric form of Red Fluorescent Protein | <p>mRFP is a monomeric form of Red Fluorescent Protein | ||
<p><img class="border" src="https://static.igem.org/mediawiki/2011/b/b1/ICL_RFP_Culture.png" width="300px" /></p> | <p><img class="border" src="https://static.igem.org/mediawiki/2011/b/b1/ICL_RFP_Culture.png" width="300px" /></p> | ||
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Revision as of 12:36, 16 September 2011
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.
Dendra 2
Dendra 2 is a green fluorescent protein that is capable of being irreversibly photoconverted by single-photon stimulation from excitation at 486nm and emission at 505nm wavelength to 558nm excitation and 575nm emission wavelength. This means that its natural state is similar to GFP, but upon photoconversion it can be excited as RFP. Photoconversion can occur using two wavelengths 488 and 405nm (Gurskaya et al., 2006)
Photoconversion
Video 1:A time-lapse video shows the conversion of cells in area 1. The single cell in area 3 serves as a negative control. It was not bleached by the laser and therefore continued to absorb light at a lower wavelength and emit green fluorescence.
Video 2:A video of another photoconversion of Dendra in E. coli cells that have been taken up into Arabidopsis roots can be seen.
How did we photoconvert Dendra 2 whilst exciting and detecting RFP?
Using a FluoroMax-3 machine, and enlisting the expertise of Tom Wilson, we used Tom's 405nm laser to photoconvert Dendra 2. This was done by aiming the laser into the sample cuvette
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.
Thermostability
GFP
mRFP
mRFP is a monomeric form of Red Fluorescent Protein
CFP
