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| − | <div style='padding-left: 20%;padding-right: 20%;text-indent: 30px;'>This year we are proud to submit a series of parts allowing for the modular control of gene expression speed, using the mf-Lon/pdt protein degradation system. These parts will enable users to predictably control the temporal dynamical behavior of their gene of choice, incorporating the speed-control system seamlessly into their circuit with our easy-to-clone parts. We include a number of variations in reporter protein and inducible promoter/respective inducer molecule, so that teams can tailor to the requirements of their unique circuit of interest. Additionally, every composite part submitted is flanked by the Unique Nucleotide Sequences (UNS’s) introduced to iGEM by W&M’s 2016 team to improve ease and reliability of cloning. </div> | + | <div style='padding-left: 20%;padding-right: 20%;text-indent: 30px;'>We are nominating our pLac mf-Lon (<a href="http://parts.igem.org/Part:BBa_K2333434"style='text-decoration: underline;'>K2333434</a>) construct for best composite part. This part was a cornerstone of our efforts to produce a modular method to alter gene expression speed, enabling us to test a wide variety of protease concentrations with ease. All of our primary characterization was done using this part, and we think that it will prove invaluable to anyone who wants to test a variety of mf-Lon concentrations without having to undergo a large number of cloning steps. As we demonstrated with our <a href="https://2017.igem.org/Team:William_and_Mary/Dynamic_Control"style='text-decoration: underline;'>IFFL</a> circuit, this part can also be used to produce fully functional circuit motifs, and as such could be used as part of a proof of concept or final implementation of other projects. We'd like to note that this part should fulfill our silver medal criteria, for more information please see our <a href="https://2017.igem.org/Team:William_and_Mary/For_Judges"style='text-decoration: underline;'>for judges</a> page. |
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| − | <div style='padding-bottom: 60px; '></div>
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| − | <div style='padding-left: 20%;padding-right: 20%;text-indent: 30px;'> <b>PUT THIS SOMEWHERE:</b> We would also like to note that due to a judging form mishap, we did not fill in the Silver Medal parts requirement with any BioBrick IDs. However, since these parts serve as both new parts and functional proofs of concept, they would be sufficient to fulfill the Silver Medal requirement, we would humbly ask that judges evaluate these parts (among others), as proof of our fulfillment of Silver Medal requirement I. More information on this can be found on our for Judges pages, as well as our other part related pages. </div>
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| − | <div style='padding-bottom: 60px; '></div>
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| − | <!------UNS PDT----->
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| − | <div class="row">
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| − | <div class="col-sm-7">
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| − | <center><div style='font-size: 22px;padding-bottom: 15px;'> Cloning-Ready Protein Degradation Tags </div></center>
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| − | <div style='padding-left: 10%;text-indent: 30px;'>We utilized a suite of 6 protein degradation tags (pdts) originally designed by [Collins et al 2014]. Each tag confers a distinct level of protein degradation; tags are lettered (A-F) in order of protease affinity, from strongest (highest level of degradation) to weakest. These parts have been made BioBrick compatible, codon-optimized for E. coli, and placed in between UNS sites, so that teams may easily insert them after their gene of choice using Gibson Assembly. They are also flanked by BsaI cut sites, for ease of use with Golden Gate Assembly. This allows future teams to efficiently adapt the speed-control system to their own parts and circuits, regardless of their preferred assembly method.</div>
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| − | <div>
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| − | <img src =""width="200px;"/>
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| | </div> | | </div> |
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| − | <center><div style='padding-left: 10%;'><img src="https://static.igem.org/mediawiki/2017/5/5f/T--William_and_Mary--circuit1.jpeg" width="550px;"/></div></center>
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| | + | <div style='padding-top: 50px;'></div> |
| | + | <figure style='padding-left: 200px;'> |
| | + | <img src='https://static.igem.org/mediawiki/2017/3/3e/T--William_and_Mary--circuit6.jpeg' height = "80%" width = "80%"/> |
| | + | <figcaption><div style='padding-left: 5px;padding-top: 15px; color: #808080; font-size: 14px;'>Figure 1: Schematic of K2333434, mf-Lon is produced in the presense of IPTG. Part is typically used on backbone psb3K3 </div></figcaption> |
| | + | </figure> |
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| | + | <div style='padding-top: 50px;'></div> |
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| − | </div>
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| − | <div class="col-sm-4">
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| | <center> | | <center> |
| − | <table style='float: right;'>
| + | <div style = 'padding-left: 0px; padding-bottom: 5px;font-size: 25px' ><b>All Composite Parts:</b></div></center> |
| − | <tr>
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| − | <th style='background-color: #BEB9C7;column-width: 70px;'><center>Part</center></th>
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| − | <th style='background-color: #BEB9C7;column-width: 400px;'><center>Description</center></th>
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| − | </tr>
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| − | <tr>
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| − | <td style='background-color: #9892A1;column-width: 70px; font-size: 15px;'><a href="http://parts.igem.org/Part:BBa_K2333401"> K2333401 </a></td>
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| − | <td style='background-color: #ECE7F2;column-width: 400px; font-size: 15px;'>Cloning Ready pdt #3A with Double Terminator</td>
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| − | </tr>
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| − | | + | |
| − | <tr>
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| − | <td style='background-color: #9892A1;column-width: 70px; font-size: 15px;'><a href="http://parts.igem.org/Part:BBa_K2333402"> K2333402 </a></td>
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| − | <td style='background-color: #DED9E5;column-width: 400px; font-size: 15px;'>Cloning Ready pdt #3B with Double Terminator</td>
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| − | </tr>
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| − | <tr>
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| − | <td style='background-color: #9892A1;column-width: 70px; font-size: 15px;'><a href="http://parts.igem.org/Part:BBa_K2333403"> K2333403 </a></td>
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| − | <td style='background-color: #ECE7F2;column-width: 400px; font-size: 15px;'>Cloning Ready pdt #3C with Double Terminator</td>
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| − | </tr>
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| − | <tr>
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| − | <td style='background-color: #9892A1;column-width: 70px; font-size: 15px;'><a href="http://parts.igem.org/Part:BBa_K2333404"> K2333404 </a></td>
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| − | <td style='background-color: #DED9E5;column-width: 400px; font-size: 15px;'>Cloning Ready pdt #3D with Double Terminator</td>
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| − | </tr>
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| − | <tr>
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| − | <td style='background-color: #9892A1;column-width: 70px; font-size: 15px;'><a href="http://parts.igem.org/Part:BBa_K2333405"> K2333405 </a></td>
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| − | <td style='background-color: #ECE7F2;column-width: 400px; font-size: 15px;'>Cloning Ready pdt #3D with Double Terminator</td>
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| − | </tr>
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| − | <tr>
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| − | <td style='background-color: #9892A1;column-width: 70px; font-size: 15px;'><a href="http://parts.igem.org/Part:BBa_K2333406"> K2333406 </a></td>
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| − | <td style='background-color: #DED9E5;column-width: 400px; font-size: 15px;'>Cloning Ready pdt #3F with Double Terminator</td>
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| − | </tr>
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| − | </table>
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| − | </center>
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| − | </div>
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| − | <div class="col-sm-1"></div>
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| − | </div> <!----out of row-->
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| − | <div style='padding-bottom: 60px; '></div>
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| − | <!------mScarlet pdt---->
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| − | <div class="row">
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| − | <div class="col-sm-7">
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| − | <center><div style='font-size: 22px;padding-bottom: 15px;'> Constitutive Tagged Reporters </div></center>
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| − | <div style='padding-left: 10%;text-indent: 30px;'>Our next collection of parts consist of a protein degradation tagged mScarlet reporter under the control of the strong constitutive promoter J23100. These parts, in combination with inducible mf-Lon protease constructs (listed below), allowed us to characterize the degradation properties of each protein degradation tag on a plasmid-based system. We successfully demonstrated distinct levels of protein degradation by each of the 6 pdt’s; see our full characterization here [HYPERLINK TO RESULTS PAGE]. We also included a tagless control construct (J23100 mScarlet with no pdt) as a comparison. In order to demonstrate that our protein degradation tags operated similarly regardless of the tagged protein, we also built and characterized analogous constructs with an sfGFP reporter; these are listed below in the “All Parts” table. mScarlet and sfGFP reporters have been codon-optomized for E. coli and feature a double stop codon. </div> | + | |
| − | <div>
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| − | <img src =""width="200px;"/>
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| − | </div>
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| − | <center><div style='padding-left: 10%;'><img src="https://static.igem.org/mediawiki/2017/b/bb/T--William_and_Mary--circuit2.jpeg" width="550px;"/></div></center>
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| − | </div>
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| − | <div class="col-sm-4">
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| − | <center>
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| − | <table style=''>
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| − | <tr>
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| − | <th style='background-color: #BEB9C7;column-width: 50px;'><center>Part</center></th>
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| − | <th style='background-color: #BEB9C7;column-width: 250px;'><center>Description</center></th>
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| − | </tr>
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| − | <tr>
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| − | <td style='background-color: #9892A1;column-width: 50px; font-size: 15px;'><a href="http://parts.igem.org/Part:BBa_K2333413"> K2333413 </a></td>
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| − | <td style='background-color: #ECE7F2;column-width: 250px; font-size: 15px;'>J23100 mScarlet-I</td>
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| − | </tr>
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| − | | + | |
| − | <tr>
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| − | <td style='background-color: #9892A1;column-width: 50px; font-size: 15px;'><a href="http://parts.igem.org/Part:BBa_K2333414"> K2333414 </a></td>
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| − | <td style='background-color: #DED9E5;column-width: 250px; font-size: 15px;'>J23100 mScarlet-I pdt #3A</td>
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| − | </tr>
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| − | | + | |
| − | <tr>
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| − | <td style='background-color: #9892A1;column-width: 50px; font-size: 15px;'><a href="http://parts.igem.org/Part:BBa_K2333415"> K2333415 </a></td>
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| − | <td style='background-color: #ECE7F2;column-width: 250px; font-size: 15px;'>J23100 mScarlet-I pdt #3B</td>
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| − | </tr>
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| − | | + | |
| − | <tr>
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| − | <td style='background-color: #9892A1;column-width: 50px; font-size: 15px;'><a href="http://parts.igem.org/Part:BBa_K2333416"> K2333416 </a></td>
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| − | <td style='background-color: #DED9E5;column-width: 250px; font-size: 15px;'>J23100 mScarlet-I pdt #3C</td>
| + | |
| − | </tr>
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| − | | + | |
| − | <tr>
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| − | <td style='background-color: #9892A1;column-width: 50px; font-size: 15px;'><a href="http://parts.igem.org/Part:BBa_K2333417"> K2333417 </a></td>
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| − | <td style='background-color: #ECE7F2;column-width: 250px; font-size: 15px;'>J23100 mScarlet-I pdt #3D</td>
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| − | </tr>
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| − | | + | |
| − | <tr>
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| − | <td style='background-color: #9892A1;column-width: 50px; font-size: 15px;'><a href="http://parts.igem.org/Part:BBa_K2333418"> K2333418 </a></td>
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| − | <td style='background-color: #DED9E5;column-width: 250px; font-size: 15px;'>J23100 mScarlet-I pdt #3E</td>
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| − | </tr>
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| − | <tr>
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| − | <td style='background-color: #9892A1;column-width: 50px; font-size: 15px;'><a href="http://parts.igem.org/Part:BBa_K2333419"> K2333419 </a></td>
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| − | <td style='background-color: #DED9E5;column-width: 250px; font-size: 15px;'>J23100 mScarlet-I pdt #3F</td>
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| − | </tr>
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| − | </table>
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| − | </center>
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| − | </div>
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| − | <div class="col-sm-1"></div>
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| − | </div> <!----out of row-->
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| − | <div style='padding-bottom: 60px; '></div>
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| − | <!------inducible mScarlett pdt---->
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| − | <div class="col-sm-7">
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| − | <center><div style='font-size: 22px;padding-bottom: 15px;'> Inducible Tagged Reporters </div></center>
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| − | <div style='padding-left: 10%;'>We further include a collection of 6 aTc-inducible mScarlet-I reporter constructs tagged with each respective protein degradation tag (and a pdt-less inducible mScarlet-I control construct), all under the control of the pTet promoter. This collection of parts was used in our gene expression speed measurements, allowing us to control the initiation of reporter expression using the small molecule aTc. We used these constructs along with the IPTG-inducible mf-Lon protease (listed directly below) to demonstrate distinct levels of speed to steady state in reporter expression proportional to the relative strength of each pdt; the full results of these experiments are found here (HYPERLINK TO RESULTS PAGE). We also took advantage of the inducible nature of these constructs by manipulating levels of aTc exposure in order to adjust final steady state values independently of speed control; the details of our readjustment experiments are found here (HYPERLINK TO RESULTS PAGE). Once again, we also created analogous constructs for each of these parts, replacing mScarlet with sfGFP. These constructs are listed in the All Parts table at the bottom of this page. </div>
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| − | <div>
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| − | <img src =""width="200px;"/>
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| − | </div>
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| − | <center><div style='padding-left: 10%;'><img src="https://static.igem.org/mediawiki/2017/1/13/T--William_and_Mary--circuit3.jpeg" width="550px;"/></div></center>
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| − | </div>
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| − | <center>
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| − | <table style=''>
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| − | <tr>
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| − | <th style='background-color: #BEB9C7;column-width: 50px;'><center>Part</center></th>
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| − | <th style='background-color: #BEB9C7;column-width: 250px;'><center>Description</center></th>
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| − | </tr>
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| − | | + | |
| − | <tr>
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| − | <td style='background-color: #9892A1;column-width: 50px; font-size: 15px;'><a href="http://parts.igem.org/Part:BBa_K2333427"> K2333427 </a></td>
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| − | <td style='background-color: #ECE7F2;column-width: 250px; font-size: 15px;'>UNS pTet mScarlet-I</td>
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| − | </tr>
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| − | | + | |
| − | <tr>
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| − | <td style='background-color: #9892A1;column-width: 50px; font-size: 15px;'><a href="http://parts.igem.org/Part:BBa_K2333428"> K2333428 </a></td>
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| − | <td style='background-color: #DED9E5;column-width: 250px; font-size: 15px;'>UNS pTet mScarlet-I pdt #3A</td>
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| − | </tr>
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| − | <tr>
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| − | <td style='background-color: #9892A1;column-width: 50px; font-size: 15px;'><a href="http://parts.igem.org/Part:BBa_K2333429"> K2333429 </a></td>
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| − | <td style='background-color: #ECE7F2;column-width: 250px; font-size: 15px;'>UNS pTet mScarlet-I pdt #3B</td>
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| − | </tr>
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| − | | + | |
| − | <tr>
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| − | <td style='background-color: #9892A1;column-width: 50px; font-size: 15px;'><a href="http://parts.igem.org/Part:BBa_K2333430"> K2333430 </a></td>
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| − | <td style='background-color: #DED9E5;column-width: 250px; font-size: 15px;'>UNS pTet mScarlet-I pdt #3C</td>
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| − | </tr>
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| − | | + | |
| − | <tr>
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| − | <td style='background-color: #9892A1;column-width: 50px; font-size: 15px;'><a href="http://parts.igem.org/Part:BBa_K2333431"> K2333431 </a></td>
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| − | <td style='background-color: #ECE7F2;column-width: 250px; font-size: 15px;'>UNS pTet mScarlet-I pdt #3D</td>
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| − | </tr>
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| − | | + | |
| − | <tr>
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| − | <td style='background-color: #9892A1;column-width: 50px; font-size: 15px;'><a href="http://parts.igem.org/Part:BBa_K2333432"> K2333432 </a></td>
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| − | <td style='background-color: #DED9E5;column-width: 250px; font-size: 15px;'>UNS pTet mScarlet-I pdt #3E</td>
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| − | </tr>
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| − | | + | |
| − | <tr>
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| − | <td style='background-color: #9892A1;column-width: 50px; font-size: 15px;'><a href="http://parts.igem.org/Part:BBa_K2333433"> K2333433 </a></td>
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| − | <td style='background-color: #DED9E5;column-width: 250px; font-size: 15px;'>UNS pTet mScarlet-I pdt #3F</td>
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| − | </tr>
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| − | | + | |
| − | </table>
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| − | | + | |
| − | </center>
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| − | </div>
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| − | <div class="col-sm-1"></div>
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| − | </div> <!----out of row-->
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| − | <div style='padding-bottom: 60px; '></div>
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| − | <!------inducible Lon---->
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| − | <div class="row">
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| − | <div class="col-sm-7">
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| − | <center><div style='font-size: 22px;padding-bottom: 15px;'> Inducible mf-Lon Protease </div></center>
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| − | | + | |
| − | <div style='padding-left: 10%;'>Our gene expression speed control system features the E. coli-orthogonal mf-Lon protease originally characterized by [sauer citation] , which specifically targets the above protein degradation tags with varying affinities corresponding to varying degradation rates. We have modified the mf-Lon gene via codon-optimization for iGEM use and added a double terminator (for details see [link basic part]). We have submitted mf-Lon constructs that are inducible by IPTG and arabinose, respectively. We used the IPTG-inducible mf-Lon construct in tandem with the above aTc-inducible pdt reporter constructs to obtain gene expression speed measurements--these results can be found here (HYPERLINK TO RESULTS PAGE). </div>
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| − | <div>
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| − | <img src =""width="200px;"/>
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| − | </div>
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| − | </div>
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| − | <div class="col-sm-4">
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| − | <center>
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| − | <table style=''>
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| − | <tr>
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| − | <th style='background-color: #BEB9C7;column-width: 50px;'><center>Part</center></th>
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| − | <th style='background-color: #BEB9C7;column-width: 200px;'><center>Description</center></th>
| + | |
| − | </tr>
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| − | | + | |
| − | <tr>
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| − | <td style='background-color: #9892A1;column-width: 50px; font-size: 15px;'><a href="http://parts.igem.org/Part:BBa_K2333434"> K2333434 </a></td>
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| − | <td style='background-color: #ECE7F2;column-width: 200px; font-size: 15px;'>pLac0-1 mf-Lon</td>
| + | |
| − | </tr>
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| − | | + | |
| − | <tr>
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| − | <td style='background-color: #9892A1;column-width: 50px; font-size: 15px;'><a href="http://parts.igem.org/Part:BBa_K2333435"> K2333435 </a></td>
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| − | <td style='background-color: #DED9E5;column-width: 200px; font-size: 15px;'>pBad mf-Lon</td>
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| − | <center><img src="https://static.igem.org/mediawiki/2017/b/b0/T--William_and_Mary--circuit5.jpeg" width="400px"></center></div>
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| − | <center><img src="https://static.igem.org/mediawiki/2017/3/3e/T--William_and_Mary--circuit6.jpeg" width="550px"></center></div>
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| − | <div style='padding-bottom: 60px; '></div>
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| − | <!------Maryland Parts---->
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| − | <div class="col-sm-7">
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| − | <center><div style='font-size: 22px;padding-bottom: 15px;'> Degradation-tagged Copper Sensor </div></center>
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| − | <div style='padding-left: 10%;'>As part of our collaboration with the University of Maryland iGEM team, we built 6 additional constructs incorporating our protein degradation tags onto their CueR-based copper sensor (link to UMD copper sensor part page??). These parts allowed us to demonstrate persistence of speed-change effects for protein outputs beyond simple reporter proteins, and provide an example of a practical application of our speed-control system to improve biosensor output speed. The details of these experiments can be found here (HYPERLINK TO RESULTS PAGE). </div>
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| − | <center><div style='padding-left: 10%;'><img src="https://static.igem.org/mediawiki/2017/b/bc/T--William_and_Mary--circuit4.jpeg" width="550px;"/></div></center>
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| − | <th style='background-color: #BEB9C7;column-width: 50px;'><center>Part</center></th>
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| − | <th style='background-color: #BEB9C7;column-width: 250px;'><center>Description</center></th>
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| − | <td style='background-color: #9892A1;column-width: 50px; font-size: 15px;'><a href="http://parts.igem.org/Part:BBa_K2333437"> K2333437 </a></td>
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| − | <td style='background-color: #DED9E5;column-width: 250px; font-size: 15px;'>Copper Sensor pdt #3A</td>
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| − | </tr>
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| − | <td style='background-color: #9892A1;column-width: 50px; font-size: 15px;'><a href="http://parts.igem.org/Part:BBa_K2333438"> K2333438 </a></td>
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| − | <td style='background-color: #ECE7F2;column-width: 250px; font-size: 15px;'>Copper Sensor pdt #3B</td>
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| − | </tr>
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| − | <td style='background-color: #9892A1;column-width: 50px; font-size: 15px;'><a href="http://parts.igem.org/Part:BBa_K2333439"> K2333439 </a></td>
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| − | <td style='background-color: #DED9E5;column-width: 250px; font-size: 15px;'>Copper Sensor pdt #3C</td>
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| − | </tr>
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| − | <td style='background-color: #9892A1;column-width: 50px; font-size: 15px;'><a href="http://parts.igem.org/Part:BBa_K2333440"> K2333440 </a></td>
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| − | <td style='background-color: #ECE7F2;column-width: 250px; font-size: 15px;'>Copper Sensor pdt #3D</td>
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| − | </tr>
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| − | <td style='background-color: #9892A1;column-width: 50px; font-size: 15px;'><a href="http://parts.igem.org/Part:BBa_K2333441"> K2333441 </a></td>
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| − | <td style='background-color: #DED9E5;column-width: 250px; font-size: 15px;'>Copper Sensor pdt #3E</td>
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| − | </tr>
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| − | <td style='background-color: #9892A1;column-width: 50px; font-size: 15px;'><a href="http://parts.igem.org/Part:BBa_K2333442"> K2333442 </a></td>
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| − | <td style='background-color: #DED9E5;column-width: 250px; font-size: 15px;'>Copper Sensor pdt #3F</td>
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| − | </tr>
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| − | </table>
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| − | </center>
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| − | <div style='padding-bottom: 40px; '></div>
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| − | <!-----All Parts---->
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| − | <center><div style=' padding-bottom: 20px; font-size: 22px; '> All Submitted Parts</div ></center> | + | <div style='background: #808080; margin: 0px 190px 20px 190px; height:1px;></div> |
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| | <td style='background-color: #DED9E5;column-width: 250px; font-size: 15px;'>J23100 mScarlet-I pdt #3F</td> | | <td style='background-color: #DED9E5;column-width: 250px; font-size: 15px;'>J23100 mScarlet-I pdt #3F</td> |
| | </tr> | | </tr> |
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