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| <div class="post-content"><p style="font-size:17px;" align="justify"> | | <div class="post-content"><p style="font-size:17px;" align="justify"> |
− | <p style="font-size:17px"; align="justify"> | + | <p style="font-size:17px"; align="justify">Finally, we tested the ability of our OnePot PURE system to express super folding GFP. |
− | The DNA sequence coding for catechol-2,3-deoxygenase (CDO), and completed with an ribosome binding site (rbs) and T7 promoter and terminator sites, was successfully assembled from the XylE (gene coding for CDO) template provided in the iGEM 2019 DNA Distribution kit, by using a 2-step PCR protocol.
| + | We initiated 10μl reactions using our own Protein Solution, Energy Solution and Ribosome Solution, while altering the concentration of the DNA template. The goal of this experiment was to determine the optimal concentration of DNA in each reaction. In addition, we included similar reactions of PURExpress from NEB to compare our system to the commercially available one. |
− | The gene assembly was verified by a Sanger DNA sequencing which showed that the DNA template was 99.8% accurate, for a total sequence length of 1045 bases. | + | In the following graphs we show the Fluorecein concentration of the samples, measured at an excitation wavelength of 535 nm, using 2.5, 5 and 10nM of DNA template.</br> </br> |
− | </p>
| + | </p> |
− | <p style="font-size:17px"; align="justify">
| + | |
− | This sequence was then expressed in our OnePot PURE cell-free system and incubated in presence of catechol.
| + | |
− | A yellow color was observed after 30 minutes of incubation, and it became brighter one
| + | <center> |
− | hour after the start of the reaction. There were no colors in the t wo controls performed, one without catechol but with CDO template and the other one without CDO template but with catechol. This proved that the color was indeed created by the reaction of CDO with catechol and not by self-oxidation of catechol.
| + | <img src="https://static.igem.org/mediawiki/2019/b/b2/T--EPFL--resultsOnePot10.png" > |
− | </p>
| + | |
− | <center>
| + | </center> |
− | <img src="https://static.igem.org/mediawiki/2019/9/9d/T--EPFL--Detection_cdo_result.jpg" >
| + | <center> |
− | </center>
| + | <img src="https://static.igem.org/mediawiki/2019/7/75/T--EPFL--resultsOnePot5.png" > |
− | </div> | + | |
| + | </center> |
| + | <center> |
| + | <img src="https://static.igem.org/mediawiki/2019/e/e7/T--EPFL--resultsOnePot25.png" > |
| + | |
| + | </center> |
| + | <center> |
| + | <img src="https://static.igem.org/mediawiki/2019/7/70/T--EPFL--resultsOnePot.png" > |
| + | |
| + | </center> |
| + | |
| + | <div class="post-content"><p style="font-size:17px;"align="justify"> As we can see, OnePot PURE has an even higher expression of sfGFP than PURExpress*. The initial hypothesis, that 5nM of DNA is the optimal concentration, is also confirmed. We still get slightly higher fluorescence at 10nM but the difference is insignificant when taking into account that the concentration of DNA in our sample doubled. </br> </br> |
| + | |
| + | *Because of the extreme difference between the output of OnePot and PURExpress, we repeated the experiments to rule out the effect of a human error using a different batch of PURExpress, but we got the exact same results.</br> </br> |
| + | </p> |
| | | |
| </p> | | </p> |