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| − | <h2 style='padding-top: 40px; font-family: "Verlag-Book"; font-size: 50px;'>
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| − | Measurement
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| − | <p class='large' style="padding-left:40px; text-indent: 50px;">
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| − | We performed a number of rigorous and thorough measurements, on each component of the circuit control toolbox. We measured the effect of the insulating ribozyme <a href= "https://2016.igem.org/Team:William_and_Mary/RiboJ"> RiboJ</a> on relative fluorescence, and confirmed that it does indeed insulate proteins from genetic context. <b>(Figure 1)</b>. We also measured RiboJ’s effect on absolute expression, and discovered that it increases absolute expression <b> (Figure 2)</b>. We created a RiboJ promoter library containing the Anderson Library of Promoters as well as additional inducible promoters, this library will form the basis for an ongoing rigorous characterization of the effect of RiboJ on absolute fluorescence.
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| − | <p class='large' style="padding-left:40px; text-indent: 50px;">
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| − | We also characterized the community collection of <a href= "https://2016.igem.org/Team:William_and_Mary/RBS">RBSs</a> under the inducible promoter pLacO-1 <b>(Figure 3)</b>. While our characterization was mostly in line with existing values, it highlighted the need to characterize over a range of conditions rather than at just one level of expression. Since our characterization was insulated using RiboJ, it should be applicable in all genetic contexts, even though past characterizations may not be. Additionally, we collaborated with UPitt and Alverno to measure our RBS characterization parts in cell free systems <b>(Figure 4 and 5)</b>. Characterization was also performed on the <a href= "https://2016.igem.org/Team:William_and_Mary/Interlab>">Interlab </a> parts using FACS.
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| − | <p class='large' style="padding-left:40px; text-indent: 50px;">
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| − | Finally, we performed the first iGEM characterization of two new parts, which allow for orthogonal control over the output of a given arbitrary circuit. We replicated and characterized both the original and our improved <a href= "https://2016.igem.org/Team:William_and_Mary/Synthetic_Enhancer"> synthetic enhancer </a> on the Biobrick Backbone, which allows for multistate output beyond a normal transfer function <b>(Figures 6-7)</b>. We also performed characterization of the effect of <a href= "https://2016.igem.org/Team:William_and_Mary/Binding_Array">molecular titration</a> on transfer functions by using a TetO array to cause a leftward shift <b>(Figure 8)</b>. A <a href= "https://2016.igem.org/Team:William_and_Mary/Model">model</a> was made based on literature values, and our characterization matched our models prediction <b>(Figure 9)</b>.
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| − | <img src="https://static.igem.org/mediawiki/2016/9/95/T--William_and_Mary--RiboJ-Convergencea.svg" width="800px">
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| − | <p class='large' style="padding-left:40px; text-indent: 50px; color:#A9A9A9; font-size: .75em !important;">
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| − | <b>Figure 1A</b>
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| − | <img src="https://static.igem.org/mediawiki/2016/1/10/T--William_and_Mary--RiboJ-ConvergenceB.svg" width="800px">
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| − | </p>
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| − | <p class='large' style="padding-left:40px; text-indent: 50px; color:#A9A9A9; font-size: .75em !important;">
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| − | <b>Figure 1B</b>
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| − | <p class='large' style="padding-left:40px; text-indent: 50px; color:#A9A9A9; font-size: .75em !important;">
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| − | <b>Figure 1A and B:</b> RiboJ insulates transfer functions from genetic context, collapsing the relative transfer functions of two different reporters. </p>
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| − | <img src="https://static.igem.org/mediawiki/2016/0/04/T--William_and_Mary--RiboJ-abs-A.svg" width="800px">
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| − | <p class='large' style="padding-left:40px; text-indent: 50px; color:#A9A9A9; font-size: .75em !important;">
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| − | <b>Figure 2A</b>
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| − | <img src="https://static.igem.org/mediawiki/2016/e/e3/T--William_and_Mary--RiboJ-abs-B.svg" width="800px">
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| − | </p>
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| − | <p class='large' style="padding-left:40px; text-indent: 50px; color:#A9A9A9; font-size: .75em !important;">
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| − | <b>Figure 2B</b>
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| − | <p class='large' style="padding-left:40px; text-indent: 50px; color:#A9A9A9; font-size: .75em !important;">
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| − | <b>Figure 2A and B:</b> RiboJ has divergent effects on absolute expression on different promoters. RiboJ decreases absolute fluorescence under the promoter pLacO-1(A), but increases it under pTac (B). </p>
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| − | <img src="https://static.igem.org/mediawiki/2016/f/f0/T--William_and_Mary--Rbs-plot.svg" width="800px">
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| − | </p>
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| − | <p class='large' style="padding-left:40px; text-indent: 50px; color:#A9A9A9; font-size: .75em !important;">
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| − | <p class='large' style="padding-left:40px; text-indent: 50px; color:#A9A9A9; font-size: .75em !important;">
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| − | <b>Figure 3:</b> Relative sfGFP characterization of the Community Collection of RBSs with RiboJ using the inducible promter pLacO-1. </p>
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| − | <img src="https://static.igem.org/mediawiki/2016/5/58/T--William_and_Mary--Rbs-5.png" width="800px">
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| − | <p class='large' style="padding-left:40px; text-indent: 50px; color:#A9A9A9; font-size: .75em !important;">
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| − | <b>Figure 4:</b> Characterization (blinded) of our RBS library by University of Pittsburgh in their cell free system S30. </p>
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| − | <img src="https://static.igem.org/mediawiki/2016/b/b6/T--William_and_Mary--RBS-F6a-V1-.png" width="800px">
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| − | <p class='large' style="padding-left:40px; text-indent: 50px; color:#A9A9A9; font-size: .75em !important;">
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| − | <b>Figure 5:</b> Characterization (blinded) of our RBS library by Team Alverno in their cell free system TX-TL.
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| − | </p>
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| − | <img src="https://static.igem.org/mediawiki/2016/7/70/T-William_and_Mary--SE_Fig5.PNG" width="800px">
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| − | <p class='large' style="padding-left:40px; text-indent: 50px; color:#A9A9A9; font-size: .75em !important;">
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| − | <b>Figure 6:</b> Characterization of improved synthetic enhancer in Biobrick Backbone.
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| − | </p>
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| − | <img src="https://static.igem.org/mediawiki/2016/1/1a/T--William_and_Mary--Updated_synthetic_enhancer_overlay.svg" width="800px">
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| − | </p>
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| − | <p class='large' style="padding-left:40px; text-indent: 50px; color:#A9A9A9; font-size: .75em !important;">
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| − | <b>Figure 7:</b> Comparison of characterization of original synthetic enhancer from Amit et al. 2012 and improved synthetic enhancer on Biobrick Backbone.
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| − | </p>
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| − | <img src="https://static.igem.org/mediawiki/2016/d/d8/T--William_and_Mary--tetO_array_poster.png" width="800px">
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| − | </p>
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| − | <p class='large' style="padding-left:40px; text-indent: 50px; color:#A9A9A9; font-size: .75em !important;">
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| − | <b>Figure 8:</b> Effect of repeat TetO array on the induction curve of pTet GFP
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| − | <h3 style='padding-top: 50px; padding-bottom: 50px;'>FACS Plots</h3>
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| − | <p class='large' style="padding-left:40px; text-indent: 50px; font-size: .75em !important;">RBS B0029 1</p>
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| − | <p style="text-align: center; padding-top: 30px; padding-bottom: 30px;"><img src="https://static.igem.org/mediawiki/2016/9/93/T--William_and_Mary--R-31-1-1000.png" width="800px"></p>
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| − | <p style="text-align: center; padding-top: 30px; padding-bottom: 30px;"><img src="https://static.igem.org/mediawiki/2016/3/36/T--William_and_Mary--R-31-1-10000.png" width="800px"></p>
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| − | <p class='large' style="padding-left:40px; text-indent: 50px; font-size: .75em !important;">RBS B0031 2</p>
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| − | <p style="text-align: center; padding-top: 30px; padding-bottom: 30px;"><img src="https://static.igem.org/mediawiki/2016/2/22/T--William_and_Mary--R-31-2-0.png" width="800px"></p>
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| − | <p style="text-align: center; padding-top: 30px; padding-bottom: 30px;"><img src="https://static.igem.org/mediawiki/2016/f/fd/T--William_and_Mary--R-31-2-1.png" width="800px"></p>
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| − | <p style="text-align: center; padding-top: 30px; padding-bottom: 30px;"><img src="https://static.igem.org/mediawiki/2016/e/e7/T--William_and_Mary--R-31-2-10.png" width="800px"></p>
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| − | <p style="text-align: center; padding-top: 30px; padding-bottom: 30px;"><img src="https://static.igem.org/mediawiki/2016/7/76/T--William_and_Mary--R-31-2-100.png" width="800px"></p>
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| − | <p style="text-align: center; padding-top: 30px; padding-bottom: 30px;"><img src="https://static.igem.org/mediawiki/2016/7/71/T--William_and_Mary--R-31-2-1000.png" width="800px"></p>
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| − | <p style="text-align: center; padding-top: 30px; padding-bottom: 30px;"><img src="https://static.igem.org/mediawiki/2016/e/e5/T--William_and_Mary--R-31-2-10000.png" width="800px"></p>
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| − | <p class='large' style="padding-left:40px; text-indent: 50px; font-size: .75em !important;">RBS B0031 3</p>
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| − | <p style="text-align: center; padding-top: 30px; padding-bottom: 30px;"><img src="https://static.igem.org/mediawiki/2016/c/c2/T--William_and_Mary--R-31-3-0.png" width="800px"></p>
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| − | <p style="text-align: center; padding-top: 30px; padding-bottom: 30px;"><img src="https://static.igem.org/mediawiki/2016/f/f8/T--William_and_Mary--R-31-3-1.png" width="800px"></p>
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| − | <p style="text-align: center; padding-top: 30px; padding-bottom: 30px;"><img src="https://static.igem.org/mediawiki/2016/8/8a/T--William_and_Mary--R-31-3-10.png" width="800px"></p>
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| − | <p style="text-align: center; padding-top: 30px; padding-bottom: 30px;"><img src="https://static.igem.org/mediawiki/2016/6/6b/T--William_and_Mary--R-31-3-100.png" width="800px"></p>
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| − | <p style="text-align: center; padding-top: 30px; padding-bottom: 30px;"><img src="https://static.igem.org/mediawiki/2016/d/da/T--William_and_Mary--R-31-3-1000.png" width="800px"></p>
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| − | <p style="text-align: center; padding-top: 30px; padding-bottom: 30px;"><img src="https://static.igem.org/mediawiki/2016/8/8a/T--William_and_Mary--R-31-3-10000.png" width="800px"></p>
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| − | <p class='large' style="padding-left:40px; text-indent: 50px; font-size: .75em !important;">RBS B0032 1</p>
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| − | <p style="text-align: center; padding-top: 30px; padding-bottom: 30px;"><img src="https://static.igem.org/mediawiki/2016/5/5a/T--William_and_Mary--R-32-1-0.png" width="800px"></p>
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| − | <p style="text-align: center; padding-top: 30px; padding-bottom: 30px;"><img src="https://static.igem.org/mediawiki/2016/b/b1/T--William_and_Mary--R-32-1-1.png" width="800px"></p>
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| − | <p style="text-align: center; padding-top: 30px; padding-bottom: 30px;"><img src="https://static.igem.org/mediawiki/2016/f/f4/T--William_and_Mary--R-32-1-10.png" width="800px"></p>
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| − | <p style="text-align: center; padding-top: 30px; padding-bottom: 30px;"><img src="https://static.igem.org/mediawiki/2016/9/92/T--William_and_Mary--R-32-1-100.png" width="800px"></p>
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| − | <p style="text-align: center; padding-top: 30px; padding-bottom: 30px;"><img src="https://static.igem.org/mediawiki/2016/8/87/T--William_and_Mary--R-32-1-1000.png" width="800px"></p>
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| − | <p style="text-align: center; padding-top: 30px; padding-bottom: 30px;"><img src="https://static.igem.org/mediawiki/2016/2/2d/T--William_and_Mary--R-32-1-10000.png" width="800px"></p>
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| − | <p class='large' style="padding-left:40px; text-indent: 50px; font-size: .75em !important;">RBS B0032 1</p>
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| − | <p style="text-align: center; padding-top: 30px; padding-bottom: 30px;"><img src="https://static.igem.org/mediawiki/2016/5/5a/T--William_and_Mary--R-32-1-0.png" width="800px"></p>
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| − | <p style="text-align: center; padding-top: 30px; padding-bottom: 30px;"><img src="https://static.igem.org/mediawiki/2016/b/b1/T--William_and_Mary--R-32-1-1.png" width="800px"></p>
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| − | <p style="text-align: center; padding-top: 30px; padding-bottom: 30px;"><img src="https://static.igem.org/mediawiki/2016/f/f4/T--William_and_Mary--R-32-1-10.png" width="800px"></p>
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| − | <p style="text-align: center; padding-top: 30px; padding-bottom: 30px;"><img src="https://static.igem.org/mediawiki/2016/9/92/T--William_and_Mary--R-32-1-100.png" width="800px"></p>
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| − | <p style="text-align: center; padding-top: 30px; padding-bottom: 30px;"><img src="https://static.igem.org/mediawiki/2016/8/87/T--William_and_Mary--R-32-1-1000.png" width="800px"></p>
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| − | <p style="text-align: center; padding-top: 30px; padding-bottom: 30px;"><img src="https://static.igem.org/mediawiki/2016/2/2d/T--William_and_Mary--R-32-1-10000.png" width="800px"></p>
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| − | <p class='large' style="padding-left:40px; text-indent: 50px; font-size: .75em !important;">Email igem@wm.edu if you want to see the rest of our more than 200 FACS plots!</p>
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| − | <div align="center">
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| − | <h3 style='padding-top: 50px; padding-bottom: 50px;'>References</h3>
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| − | </div>
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| − | <p class='large' style="padding-left:70px; padding-bottom: 30px;">
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| − | <b>1.</b> C. Lou, B. Stanton, Y.-J. Chen, B. Munsky, C. A. Voigt, Ribozyme-based insulator parts buffer synthetic circuits from
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| − | genetic context. Nat. Biotechnol. 30, 1137 (2012). doi:10.1038/nbt.2401 pmid:23034349
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| − | </p>
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| | {{Team:William_and_Mary/JS_SOURCE2}} | | {{Team:William_and_Mary/JS_SOURCE2}} |
| | {{Team:William_and_Mary/FOOTER_SMALL}} | | {{Team:William_and_Mary/FOOTER_SMALL}} |
