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| | <h1>Fluorescent Reporters</h1> | | <h1>Fluorescent Reporters</h1> |
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| - | <p>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.</p> | + | <p>Dendra2 is the first photoconvertible protein that has been introduced into the registry. When excited with a certain wavelength of light, the protein is permanently converted from green to red. This interesting property allows it to be used for any processes that looks at production. In our experiments, we planned to use Dendra2 in order to monitor the metabolic viability of our <i>Escherichia coli</i> once it was in the root.</p> |
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| - | <p>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 (sfGFP) that is codon-optimised for <i>E. coli</i>. These were both used as part of our imaging experiments of bacteria inside plant roots. sfGFP was also used in our soil experiments to label our bacteria so that we could later identify them.</p> | + | |
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| - | <p>sfGFP and mRFP were also used in our assembly strategy for the Gene Guard module. sfGFP was part of the construct of our CRIM plasmid, and would be used as a reporter to evaluate the level of expression of antiholin. mRFP formed part of the plasmid construct of the Gene Guard and this would be used to measure the expression of holin and endolysin. The mRFP would also be used to track the transmission of the plasmid in subsequent conjugation assays that would be carried out to test the effectiveness of the Gene Guard.</p>
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| - | <img class="border" style="border-color:#B2B2B2;" src="https://static.igem.org/mediawiki/2011/b/b1/ICL_RFP_Culture.png" width=300px/>
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| - | <p><i>One of our cultures of </i>E. coli<i> expressing mRFP.</i></p>
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| - | <img class="border" style="border-color:#B2B2B2;" src="http://farm7.static.flickr.com/6188/6144339487_bc7fec585b.jpg" width=200px/>
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| - | <p><i>Samples of sfGFP from our thermostability assay. Note the colour change as the temperature increases from left to right.</i></p>
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| - | <p>Each of the fluorescent reporters that we studied was transformed into 5-alpha competent <i>E. coli</i> cells and then grown in large batch culture overnight. The cells were then lysed and the cell debris removed, leaving the protein suspended in 20 mM Tris buffer.</p>
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| - | <p>To further characterise these parts, we have conducted a thermostability assay to determine the temperature at which these proteins denature and cease to fluoresce. The data was collected in two overlapping sets, ranging from 35°C to 66°C, and from 57°C to 96°C. This was because it was only possible to fit a maximum of eight samples into the thermocycler at once.</p>
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| - | <p>Once the data was collected by taking fluorescence readings from a 96-well plate, it was normalised to a sample of untreated cell lysate containing the fluorescent protein. This gave a value for Relative Fluorescence, which was plotted against temperature to create a scatter plot. A line of best fit was applied, and the mid point of the sigmoid region of the line was taken as the denaturation point of the protein.</p>
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| - | <p>The protocols can be found in full on our Protocols page, which can be found by clicking on the button below.</p>
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| - | <p style="text-align:right;"><a href="https://2011.igem.org/Team:Imperial_College_London/Protocols_Auxin"><img src="https://static.igem.org/mediawiki/2011/5/58/ICL_ProtocolIconDark.png" width="180px" /></a></p>
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| - | <h3>Dendra 2</h3>
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| - | <p>Dendra 2 is a green fluorescent protein that is capable of being irreversibly photo-converted by single-photon stimulation from excitation at 486 nm and emission at 505 nm wavelength to 558 nm excitation and 575nm emission wavelength. This means that its natural state is similar to GFP, but upon photo-conversion it can be excited as RFP. Photo-conversion can occur using two wavelengths 488 and 405 nm (Gurskaya et al., 2006)</p>
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| - | <h4>Photo-conversion</h4><p><img align="right" class="border" src="" width="100px" height="300px" /></p>
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| - | <p>Featured here are two of our videos demonstrating the photo-conversion of Dendra 2. Both videos show dendra expressing E.coli inside plant roots as they are photo-converted.</p>
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| | <iframe width="560" height="315" src="http://www.youtube.com/embed/dEyfjhkS-gQ?rel=0" frameborder="0" allowfullscreen></iframe> | | <iframe width="560" height="315" src="http://www.youtube.com/embed/dEyfjhkS-gQ?rel=0" frameborder="0" allowfullscreen></iframe> |
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| - | <p><i>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.</i></p>
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| - | <p><b>How did we photo-convert Dendra 2 whilst exciting and detecting RFP?</b></p>
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| | <div class="imgbox" style="width:260px; float:left;"> | | <div class="imgbox" style="width:260px; float:left;"> |
| | <img class="border" src="http://farm7.static.flickr.com/6063/6144983986_aa8dbef68d_m.jpg" width="240px" /> | | <img class="border" src="http://farm7.static.flickr.com/6063/6144983986_aa8dbef68d_m.jpg" width="240px" /> |
| | <p><i>Tim uses his expertise to assist us.</i></p> | | <p><i>Tim uses his expertise to assist us.</i></p> |
| | </div> | | </div> |
| - | <div class="imgbox" style="width:260px;float:right;"> | + | <p> Many other exciting applications such as more accurate promoter characterisation will be possible since one would be able to photoconvert all of the Dendra2 and then measure the synthesis rate from point zero at different OD's. We would have loved to flesh out this new characterization method but sadly ran out of time. However, we were able to get many interesting results.</p> |
| - | <img class="border" src="http://farm7.static.flickr.com/6070/6144433291_da918e33c4_m.jpg" width="240px" />
| + | <p>First we tested the photoconvertability of the Dendra2. In order to do that we used a FluoroMax-3 machine. Luckily, Tim Wilson was kind enough to help us set up the fluorometer in order to perform our experiments. We found that Dendra2 will fluoresce red upon conversion by light at a wavelength of 405 nm very quickly.</p> |
| - | <p><i>A look inside the fluorometer.</i></p> | + | |
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| - | <p>Using a FluoroMax-3 machine, and enlisting the expertise of Tim Wilson, we used Tim's 405 nm laser to photo-convert Dendra 2. This was done by aiming the laser into the sample cuvette from above.</p>
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| - | <p>So that we could excite Dendra 2 and measure its fluorescence as it was being photo-converted, Tim placed a filter taken from the Gel Imager in front of the detector so that we could detect the fluorescence from Dendra 2 without any interference from the excitation laser</p>
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| | <p><img align="center" class="border" src="https://static.igem.org/mediawiki/2011/9/9a/ICL_dendra_photoconversion.png" width="600px" /></p> | | <p><img align="center" class="border" src="https://static.igem.org/mediawiki/2011/9/9a/ICL_dendra_photoconversion.png" width="600px" /></p> |
| - | <p>This graph illustrates the photo-conversion of Dendra 2. The increase in fluorescence at 575 nm indicates that Dendra 2 begins to fluoresce red upon conversion by light at a wavelength of 405 nm. This also shows that the conversion is very rapid, which lends itself well to in vivo use as a fluorescent reporter.</p>
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| - | <h4>Thermostability</h4>
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| - | <p><img align="center" class="border" src="https://static.igem.org/mediawiki/2011/archive/a/a2/20110917173557!DendraCurve.png" width="600px" /></p>
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| - | <p>This graph shows that Dendra 2 has a very high denaturation point at around 88°C. This lends the Dendra 2 protein very well to reporter assays where a high temperature is required. Our data also suggests that Dendra 2 may fluoresce more brightly as it is heated.</p>
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| - | <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>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 are submitting to the registry has been codon-optimised for <i>E. coli</i>.</p>
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| - | <h4>Thermostability</h4>
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| - | <p><img align="center" class="border" src="https://static.igem.org/mediawiki/2011/b/ba/SfGFPCurve.png" width="600px" /></p>
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| - | <p>From the midpoint of the sigmoidal curve, we can find the point at which half of the protein is denatured. For sfGFP, that point occurs at 78°C.</p>
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| - | <h3>GFP</h3><p><img align="right" class="border" src="https://static.igem.org/mediawiki/2011/f/f1/ICL_GFP_PURE.png" width="100px" height="300px" /></p>
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| - | <p>This GFP expression sequence is BioBrick BBa_I13522, from the Standard Registry of Biological Parts. This is a GFP coding sequence under the control of a pTet promoter. This was taken from the registry distribution and transformed into competent <i>E. coli</i> cells.</p>
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| - | <p>This helped to form the part of our project that required the re-categorisation of existing BioBricks as we carried out a thermostability assay.</p>
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| - | <h4>Thermostability</h4>
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| - | <p><img align="center" class="border" src="https://static.igem.org/mediawiki/2011/e/e6/RealGFPCurve.png" width="600px" /></p>
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| - | <h3>mRFP</h3><p><img align="right" class="border" src="https://static.igem.org/mediawiki/2011/7/7a/ICL_RFP_PURE.png" width="100px" height="300px" /></p>
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| - | <p>mRFP is a monomeric form of Red Fluorescent Protein, and the expression sequence that we used to obtain our samples is BioBrick BBa_I13521 from the Registry. This is a composite part made up of the coding sequence for mRFP under the control of a pTet promoter. This was taken from the registry distribution and transformed into competent <i>E. coli</i> cells.</p>
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| - | <p>We carried out a thermostability assay on mRFP in order to add the data to the registry data sheet.</p>
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| - | <h4>Thermostability</h4>
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| - | <p><img align="center" class="border" src="https://static.igem.org/mediawiki/2011/7/71/RFPCurve.png" width="600px" /></p>
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| | + | <h2>Thermostability assay</h2> |
| | + | <p>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.</p> |
| | + | <p>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 (sfGFP) that is codon-optimised for <i>E. coli</i>. These were both used as part of our imaging experiments of bacteria inside plant roots. sfGFP was also used in our soil experiments to label our bacteria so that we could later identify them.</p> |
| | + | <p>sfGFP and mRFP were also used in our assembly strategy for the Gene Guard module. sfGFP was part of the construct of our CRIM plasmid, and would be used as a reporter to evaluate the level of expression of anti-holin. mRFP formed part of the plasmid construct of the Gene Guard and this would be used to measure the expression of holin and endolysin. The mRFP would also be used to track the transmission of the plasmid in subsequent conjugation assays that would be carried out to test the effectiveness of the Gene Guard.</p> |
| | + | <p>Since we ended up working with so many fluorescent reporters during our project, we decided to further characterize these important BioBricks. An important characteristic that has been omitted from the registry page is the thermostability of these proteins. This is an important aspect when choosing a fluorescent protein for a thermophile.</p> |
| | + | <<INSERT GRAPH HERE>> |
| | </body> | | </body> |
| | </html> | | </html> |
Dendra2 is the first photoconvertible protein that has been introduced into the registry. When excited with a certain wavelength of light, the protein is permanently converted from green to red. This interesting property allows it to be used for any processes that looks at production. In our experiments, we planned to use Dendra2 in order to monitor the metabolic viability of our Escherichia coli once it was in the root.
Many other exciting applications such as more accurate promoter characterisation will be possible since one would be able to photoconvert all of the Dendra2 and then measure the synthesis rate from point zero at different OD's. We would have loved to flesh out this new characterization method but sadly ran out of time. However, we were able to get many interesting results.
First we tested the photoconvertability of the Dendra2. In order to do that we used a FluoroMax-3 machine. Luckily, Tim Wilson was kind enough to help us set up the fluorometer in order to perform our experiments. We found that Dendra2 will fluoresce red upon conversion by light at a wavelength of 405 nm very quickly.
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 (sfGFP) that is codon-optimised for E. coli. These were both used as part of our imaging experiments of bacteria inside plant roots. sfGFP was also used in our soil experiments to label our bacteria so that we could later identify them.
sfGFP and mRFP were also used in our assembly strategy for the Gene Guard module. sfGFP was part of the construct of our CRIM plasmid, and would be used as a reporter to evaluate the level of expression of anti-holin. mRFP formed part of the plasmid construct of the Gene Guard and this would be used to measure the expression of holin and endolysin. The mRFP would also be used to track the transmission of the plasmid in subsequent conjugation assays that would be carried out to test the effectiveness of the Gene Guard.
Since we ended up working with so many fluorescent reporters during our project, we decided to further characterize these important BioBricks. An important characteristic that has been omitted from the registry page is the thermostability of these proteins. This is an important aspect when choosing a fluorescent protein for a thermophile.
"
