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− | </style>
| + | #top_menu_inside, #top_menu_inside:before, #top_menu_inside:after { -webkit-box-sizing: content-box; -moz-box-sizing: border-box; box-sizing: content-box;} |
| + | #top_menu_under{font-size: 12px;} |
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| + | #top_menu_inside li{font-size: 12px;} |
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| <div class="navbar-collapse collapse" style="padding-top:5px;padding-bottom:0"> | | <div class="navbar-collapse collapse" style="padding-top:5px;padding-bottom:0"> |
| <!-- Start Navigation List --> | | <!-- Start Navigation List --> |
− | <ul class="nav navbar-nav navbar-right " style="padding-bottom:15px;height:88px;padding-top:10px"> | + | <ul class="nav navbar-nav navbar-right " style="padding-bottom:15px;padding-top:10px"> |
| <li> | | <li> |
| <a href="https://2015.igem.org/Team:Peking/JudgingCriteria">Achievements</a> | | <a href="https://2015.igem.org/Team:Peking/JudgingCriteria">Achievements</a> |
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| <div class="row"> | | <div class="row"> |
| <div class="col-md-6"> | | <div class="col-md-6"> |
− | <h2 style="font-size:20px; margin-bottom:5px; padding-bottom:0">Modeling</h2> | + | <h2 style="font-size:20px; margin-bottom:5px; padding-bottom:0">M<span style="text-transform:lowercase">odeling</span></h2> |
− | <p style="margin-top:0px;font-size:14px">The purpose of models is not to fit the data but to sharpen the questions. </p> | + | <p style="margin-top:0px;font-size:14px" id="Modeling-Overview">The purpose of models is not to fit the data but to sharpen the questions. </p> |
| </div> | | </div> |
| <div class="col-md-6"> | | <div class="col-md-6"> |
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| <!-- Sidebar for windows > 1024px --> | | <!-- Sidebar for windows > 1024px --> |
| <div id="sidebar1"class="widget widget-categories"> | | <div id="sidebar1"class="widget widget-categories"> |
− | <h4 style="font-size:18px">Marker Finder<span class="head-line"></span></h4> | + | <h4 style="font-size:18px">M<span style="text-transform:lowercase">arker Finder</span><span class="head-line"></span></h4> |
| <ul> | | <ul> |
| <li><a href="#Modeling-Overview">Overview</a></li> | | <li><a href="#Modeling-Overview">Overview</a></li> |
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| <!-- Sidebar for windows < 1024px --> | | <!-- Sidebar for windows < 1024px --> |
| <div id="sidebar2"class="widget widget-categories"> | | <div id="sidebar2"class="widget widget-categories"> |
− | <h4 style="font-size:18px">Marker Finder<span class="head-line"></span></h4> | + | <h4 style="font-size:18px">M<span style="text-transform:lowercase">arker Finder</span><span class="head-line"></span></h4> |
| <ul> | | <ul> |
| <li><a href="#Modeling-Overview">Overview</a></li> | | <li><a href="#Modeling-Overview">Overview</a></li> |
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| <!-- Page Content --> | | <!-- Page Content --> |
− | <div class="col-md-9 page-content"> | + | <div class="col-md-9 page-content" style="text-align:justify;"> |
| <div id="Modeling-Marker Finder"> | | <div id="Modeling-Marker Finder"> |
| <!-- Classic Heading --> | | <!-- Classic Heading --> |
− | <div id="Modeling-Overview" class="col-md-12" style="padding:0"> | + | <div class="col-md-12" style="padding:0"> |
| <h3 class="classic-title" style='margin-top:50px'><span>Overview</span></h3> | | <h3 class="classic-title" style='margin-top:50px'><span>Overview</span></h3> |
| <!-- Some Text --> | | <!-- Some Text --> |
− | <p>To increase the accuracy and specificity of the detection, we developed an assay over our Paired dCas9 Reporter (PC Reporter) System to get more sequence information from the target genome. The core as well as the first step of the design of the array is to screen over the entire genome and get paired specific sequences (CRISPR target sites) with high specificity as markers. <br> | + | <p>Though single marker detection using PC reporter works well in lab, in clinical diagnosis of MTB there can be more disturbance, which may mislead the diagnosis. To increase the reliability of the detection , we designed MTB multi-marker array. To select all MTB specific markers through out MTB genome and facilitate array experiment, we developed an algorithm named SSPD, which consists of 4 steps: |
− | We develop a method named SSPD to achieve our aim, which is composed of 4 steps: <br>
| + | |
| <ol style="margin-top:0;margin-bottom:0;color:#606060"> | | <ol style="margin-top:0;margin-bottom:0;color:#606060"> |
− | <li style="margin-bottom:6px">Search for guide sequences of gRNA candidates</li> | + | <li style="margin-bottom:6px">Search for all target candidates</li> |
− | <li style="margin-bottom:6px">Specificity test for each candidate</li> | + | <li style="margin-bottom:6px">Select MTB specific targets</li> |
− | <li style="margin-bottom:6px">Pair left and right target sites with optimal spacer length</li> | + | <li style="margin-bottom:6px">Pair left and right target sites as markers</li> |
− | <li style="margin-bottom:6px">Design PCR fragments</li> | + | <li style="margin-bottom:6px">Determine PCR fragments</li> |
| </ol> | | </ol> |
− | <p>We will introduce each step in detail separately with analysis about Mycobacterium tuberculosis (MTB) genome as an example. After the target sites are chosen, we developed an Oligo Generator to turn the target sites into oligonucleotides sequences for following sgRNA construction combined with our gRNA generator (Part). | + | <p>Notice that a target has the same sequence with corresponding sgRNA guide sequence, selecting MTB specific target is thus equivalent to selecting sgRNA.<br/> |
| + | In <span id="Modeling-SSPD">addition</span>, we designed Oligo Generator to generate oligo sequences from corresponding targets, together with sgRNA generator, it facilitates multiple sgRNA construction. The code can be found <a href="https://static.igem.org/mediawiki/2015/a/a6/Peking-Modeling-MarkerFinder.zip" style="color:#00afd1">here</a> with a report <a href="https://static.igem.org/mediawiki/2015/2/21/Code_Document.pdf" style="color:#00afd1">here</a>. |
| </p> | | </p> |
| </div> | | </div> |
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| <!-- Classic Heading --> | | <!-- Classic Heading --> |
− | <div id="Modeling-SSPD" class="col-md-12" style="padding:0"> | + | <div class="col-md-12" style="padding:0"> |
| <h3 class="classic-title" style='margin-top:50px'><span>SSPD Methods</span></h3> | | <h3 class="classic-title" style='margin-top:50px'><span>SSPD Methods</span></h3> |
| <!--Modeling-SSPD-S1--> | | <!--Modeling-SSPD-S1--> |
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− | <p>Recall the structure of Paired dCas9 Reporter (PC Reporter) System, a protospacer adjacent motif (PAM) sequence in the form of 5’-NGG-3’ at 3’ end of guide sequence, usually 20bp, on the non-complementary strand. (Figure 1) As it was showed in our experimental results that PAM-out orientation (5’-CCNN20-…-N20NGG-3’) was highly efficient for PC Reporter system to work, thus our model would focus on this orientation. (However, it can be more convenient to adjust our program for guide sequence design also with other orientations. See more in <a href="" style="color:#00afd1">Supplementary Information 1 </a>) | + | <p>The CRISPR/dCas9 requires a protospacer adjacent motif (PAM) sequence in the form of 5’-NGG-3’ downstream the target sequence to bind to the target. (Figure 1) Since our experimental results shows that PAM-out orientation (5’-CCN(N)<sub>20</sub>-…-(N)<sub>20</sub>NGG-3’) was highly efficient for PC Reporter system to work, our model focused on this orientation. (It is convenient to adjust our program for guide sequence design with other orientations. See more in <a href="https://static.igem.org/mediawiki/2015/a/a7/Peking-Modeling-Supplementary.pdf" style="color:#00afd1">Supplementary Information 1 </a>) |
| </p> | | </p> |
| | | |
| <div id="Modeling_Fig1"> | | <div id="Modeling_Fig1"> |
| <div class='row'> | | <div class='row'> |
− | <div class='col-md-2'></div> | + | <div class='col-md-1'></div> |
− | <div class="col-md-8"> | + | <div class="col-md-10"> |
− | <img class='col-md-8' alt="Modeling_Fig1" src="https://static.igem.org/mediawiki/2015/9/94/Peking_Modeling_Figure1.png"> | + | <img alt="Modeling_Fig1" src="https://static.igem.org/mediawiki/2015/9/94/Peking_Modeling_Figure1.png"> |
| </div> | | </div> |
− | <div class='col-md-2'></div> | + | <div class='col-md-1'></div> |
| </div> | | </div> |
| <div class="col-md-12"> | | <div class="col-md-12"> |
− | <p><b>Figure 1. Schematic illustration of guide design in PAM-out orientation.</b> Note the 20nt guide sequence is identical to target non-complementary strand.</p> | + | <p><b>Figure 1. Schematic illustration of guide design in PAM-out orientation.</b> Note that the 20nt guide sequence is identical to target.</p> |
| </div> | | </div> |
| </div> | | </div> |
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| <div id="Modeling_Table1" style="color:#606060"> | | <div id="Modeling_Table1" style="color:#606060"> |
| <p class='col-md-3'></p> | | <p class='col-md-3'></p> |
− | <table style='margin:10px;padding:10px;border-color:#606060' class='col-md-6 table'> | + | <table class='col-md-6 table table-bordered'> |
− | <tr> | + | <tr style="color:#606060"> |
− | <td>Left gRNA candidates (CCNN20)</td> | + | <td>Left gRNA candidates (CCN(N)<sub>20</sub>)</td> |
| <td>414962</td> | | <td>414962</td> |
| </tr> | | </tr> |
− | <tr> | + | <tr style="color:#606060"> |
− | <td>Right gRNA candidates (CCNN20)</td> | + | <td>Right gRNA candidates ((N)<sub>20</sub>NGG)</td> |
| <td>407371</td> | | <td>407371</td> |
| </tr> | | </tr> |
| </table> | | </table> |
− | <p class='col-md-12'><b>Table 1.</b> The number of gRNA candidates in Mycobacterium Tuberculosis genome</p> | + | <p class='col-md-12'><b>Table 1.</b> The number of gRNA candidates in <i>Mycobacterium Tuberculosis</i> genome</p> |
| </div> | | </div> |
| </div> | | </div> |
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| <p><b><large>Specificity test for each candidate</large></b></p> | | <p><b><large>Specificity test for each candidate</large></b></p> |
− | <p>Specificity of guide sequence of gRNA here is defined as the probability of the gRNA binds to the corresponding target site instead of other similar non-target sites. It is measured by taking both quantity of potential off-target sites and similarity between off-target sites and the unique target site into consideration. Since sputum sample is commonly used in MTB detection, we compared our guide sequence candidates with Human Oral Meta-Genome (HOMG), and reserved the specific sequences orthogonal to oral meta-genome to avoid false positive signals in MTB detection. Here we adopted a BLAST-based 2-step filter approach to realize it. In general, the two steps are: a) Filter out guides with off-targets that have 12 bp PAM-proximal sequence identical to corresponding target; b) Score the reserved gRNA on specificity. The principle of the score-rule is that higher specificity should get higher score (see the detail below). Thus we can easily filter out guides with high off-target probability, which is indicated by a low score. | + | <p>Specificity of guide sequence of gRNA here is defined as the probability of the gRNA binds to the corresponding target site instead of other similar non-target sites. It is measured by taking both quantity of potential off-target sites and similarity between off-target sites and the unique target site into consideration. Since we hope to take sample directly from human mouth, we compared our guide sequence candidates with Human Oral Meta-Genome (HOMG), and reserved only the orthogonal ones to avoid false positive in MTB detection. We adopted a BLAST-based 2-step filtration approach to realize it. In general, the two steps are: a) Filter out guides with off-targets that have 12 bp PAM-proximal sequence identical to corresponding target; b) Score the reserved gRNA on specificity. The principle of the score-rule is that higher specificity should get higher score (see the detail below). Thus we can easily filter out guides with high off-target probability, which is indicated by a low score. |
| </p> | | </p> |
| <div id="SSPD-filter1" class='col-md-12' style='margin:0; padding:0'> | | <div id="SSPD-filter1" class='col-md-12' style='margin:0; padding:0'> |
| <p><b>a) PAM-proximal 12bp filtration by BLAST</b></p> | | <p><b>a) PAM-proximal 12bp filtration by BLAST</b></p> |
− | <p>Previous research has demonstrated that SpCas9 tolerates mismatches to a greater extent in the PAM-distal region than the PAM-proximal region, and the PAM-proximal 8-12nt of the target largely determine the specificity of the targets. Guide sequences of gRNA whose target has identical PAM-proximal 12nt off-targets is highly possible to hit off target, which suggest its fate of being eliminated. Remaining guides are uploaded to BLAST again for potential off-target site finding, target similar sequences found by BLAST are recorded as all off-target sites for the second filtration, those ignored are considered not contributing to off-target. | + | <p>Previous research has demonstrated that SpCas9 tolerates mismatches to a greater extent in the PAM-distal region than the PAM-proximal region, and the PAM-proximal 8-12nt of the target largely determine the specificity of the targets. Guide sequences of gRNA whose target has identical PAM-proximal 12nt off-targets is highly possible to hit off target, which suggest its fate of being eliminated. Remaining guides are uploaded to BLAST again for potential off-target site finding. Sequences that are similar to target and found by BLAST are recorded as all off-target sites for the second filtration, those ignored are considered not contributing to off-target. |
| </p> | | </p> |
| <div id="Modeling_Table2" style="color:#606060"> | | <div id="Modeling_Table2" style="color:#606060"> |
| <p class='col-md-3'></p> | | <p class='col-md-3'></p> |
− | <table style='margin:10px;padding:10px;border-color:#606060' class=' table col-md-6'> | + | <table class=' table col-md-6 table-bordered'> |
− | <tr> | + | <tr style="color:#606060"> |
− | <td>Left gRNA candidates (CCNN20)</td> | + | <td>Left gRNA candidates (CCN(N)<sub>20</sub>)</td> |
| <td>55156</td> | | <td>55156</td> |
| </tr> | | </tr> |
− | <tr> | + | <tr style="color:#606060"> |
− | <td>Right gRNA candidates (CCNN20)</td> | + | <td>Right gRNA candidates ((N)<sub>20</sub>NGG)</td> |
| <td>54938</td> | | <td>54938</td> |
| </tr> | | </tr> |
| </table> | | </table> |
− | <p class='col-md-12'><b>Table 2.</b> The number of gRNA candidates left after the first filtration in Mycobacterium Tuberculosis genome</p> | + | <p class='col-md-12'><b>Table 2.</b> The number of gRNA candidates left after the first filtration in <i>Mycobacterium Tuberculosis</i> genome</p> |
| </div> | | </div> |
| </div> | | </div> |
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| <div class="row"id="fig1"> | | <div class="row"id="fig1"> |
− | <div class="col-md-1"></div> | + | <div class="col-md-2"></div> |
− | <div class="col-md-10"> | + | <div class="col-md-8"> |
| <img class='huge-formula-line' alt="Peking-Modeling-single_score.gif" src="https://static.igem.org/mediawiki/2015/e/e6/Peking-Modeling-single_score.gif"> | | <img class='huge-formula-line' alt="Peking-Modeling-single_score.gif" src="https://static.igem.org/mediawiki/2015/e/e6/Peking-Modeling-single_score.gif"> |
| </div> | | </div> |
− | <div class="col-md-1"></div> | + | <div class="col-md-2"></div> |
| </div> | | </div> |
| | | |
| <div class="row"id="fig2"> | | <div class="row"id="fig2"> |
− | <div class="col-md-1"></div>
| + | |
− | <div class="col-md-10"> | + | <div class="col-md-12"> |
| <img class='formula-inline' alt="Peking-Modeling-single_site_mismatch_list.gif" src="https://static.igem.org/mediawiki/2015/7/7e/Peking-Modeling-single_site_mismatch_list.gif"> | | <img class='formula-inline' alt="Peking-Modeling-single_site_mismatch_list.gif" src="https://static.igem.org/mediawiki/2015/7/7e/Peking-Modeling-single_site_mismatch_list.gif"> |
| </div> | | </div> |
− | <div class="col-md-1"></div> | + | |
| </div> | | </div> |
| | | |
| | | |
| <p> | | <p> |
− | In the first term, <i>e</i> runs over the mismatch positions set <img class='small-formula-inline' alt="Peking-Modeling-set_M.gif" src="https://static.igem.org/mediawiki/2015/6/6d/Peking-Modeling-set_M.gif" style="max-width:50px"> between the guide and the off-target site, with <img class='small-formula-inline' alt="Peking-Analysis-W.gif" src="https://static.igem.org/mediawiki/2015/6/6e/Peking-Analysis-W.gif" style="max-width:50px"> representing the experimentally-determined effect of mismatch position on targeting. Term two refers to the effect caused by mean pairwise distance between mismatches(<img class='formula-inline' alt="Peking-Modeling-d_mean.gif" src="https://static.igem.org/mediawiki/2015/3/32/Peking-Modeling-d_mean.gif" style="max-width:50px">). Term three represent a dampening penalty for highly mismatched off-targets, where <img class='formula-inline' alt="Peking-Modeling-n_m.gif" src="https://static.igem.org/mediawiki/2015/e/ea/Peking-Modeling-n_m.gif" style="max-width:50px"> refers to the number of mismatches. | + | In the first term, <i>e</i> runs over the mismatch positions set <img class='small-formula-inline' alt="Peking-Modeling-set_M.gif" src="https://static.igem.org/mediawiki/2015/6/6d/Peking-Modeling-set_M.gif" style="width:25px"> between the guide and the off-target site, with <img class='small-formula-inline' alt="Peking-Analysis-W.gif" src="https://static.igem.org/mediawiki/2015/6/6e/Peking-Analysis-W.gif" style="width:25px"> representing the experimentally-determined effect of mismatch position on targeting. Term two refers to the effect caused by mean pairwise distance between mismatches(<img class='formula-inline' alt="Peking-Modeling-d_mean.gif" src="https://static.igem.org/mediawiki/2015/3/32/Peking-Modeling-d_mean.gif" style="width:25px">). Term three represent a dampening penalty for highly mismatched off-targets, where <img class='formula-inline' alt="Peking-Modeling-n_m.gif" src="https://static.igem.org/mediawiki/2015/e/ea/Peking-Modeling-n_m.gif" style="width:25px"> refers to the number of mismatches. |
| </p> | | </p> |
| | | |
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| | | |
| <div class="row" id ="figgggg"> | | <div class="row" id ="figgggg"> |
− | <div class="col-md-3"></div> | + | <div class="col-md-4"></div> |
− | <div class="col-md-6"> | + | <div class="col-md-4"> |
| <img class='big-formula-line' alt="Modeling_Fm3" src="https://static.igem.org/mediawiki/2015/d/d6/Peking-Modeling-All_score.gif"> | | <img class='big-formula-line' alt="Modeling_Fm3" src="https://static.igem.org/mediawiki/2015/d/d6/Peking-Modeling-All_score.gif"> |
| </div> | | </div> |
− | <div class="col-md-3"></div> | + | <div class="col-md-4"></div> |
| </div> | | </div> |
− | <p> <i>h</i> runs over the potential off-target set <img class='small-formula-inline' alt="Peking-Modeling-set_T.gif" src="https://static.igem.org/mediawiki/2015/a/a0/Peking-Modeling-set_T.gif" style="max-width:50px"> of guide sequence </p> | + | <p> <i>h</i> runs over the potential off-target set <img class='small-formula-inline' alt="Peking-Modeling-set_T.gif" src="https://static.igem.org/mediawiki/2015/a/a0/Peking-Modeling-set_T.gif" style="width:25px"> of guide sequence </p> |
| </div> | | </div> |
| <p>A higher overall score indicates a better guide with few or weak potential off-targets. Guides with individual off-target sites score 2% or above are eliminated, the remaining are ranked in the order of overall score from 100% to 0%, with a list of off-targets presented in individual score descending order. Guides having an overall score of greater than 45% are reserved finally while others were filtered out in case their lack of specificity. | | <p>A higher overall score indicates a better guide with few or weak potential off-targets. Guides with individual off-target sites score 2% or above are eliminated, the remaining are ranked in the order of overall score from 100% to 0%, with a list of off-targets presented in individual score descending order. Guides having an overall score of greater than 45% are reserved finally while others were filtered out in case their lack of specificity. |
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| <div id="Modeling_Table3" style="color:#606060"> | | <div id="Modeling_Table3" style="color:#606060"> |
| <p class='col-md-3'></p> | | <p class='col-md-3'></p> |
− | <table style='margin:10px;padding:10px' class='col-md-6 table'> | + | <table class='col-md-6 table table-bordered'> |
| <tr> | | <tr> |
− | <td>Left gRNA candidates (CCNN20)</td> | + | <td>Left gRNA candidates (CCN(N)<sub>20</sub>)</td> |
| <td>54417</td> | | <td>54417</td> |
| </tr> | | </tr> |
| <tr> | | <tr> |
− | <td>Right gRNA candidates (CCNN20)</td> | + | <td>Right gRNA candidates ((N)<sub>20</sub>NGG)</td> |
| <td>54288</td> | | <td>54288</td> |
| </tr> | | </tr> |
| </table> | | </table> |
− | <p class='col-md-12'><b>Table 2.</b> The number of gRNA candidates left after the first filtration in Mycobacterium Tuberculosis genome</p> | + | <p class='col-md-12'><b>Table 2.</b> The number of gRNA candidates left after the first filtration in <i>Mycobacterium Tuberculosis</i> genome</p> |
| </div> | | </div> |
| <div id="Modeling_Fig3"> | | <div id="Modeling_Fig3"> |
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| <div id="Modeling_Table4" style="color:#606060"> | | <div id="Modeling_Table4" style="color:#606060"> |
| <p class='col-md-3'></p> | | <p class='col-md-3'></p> |
− | <table style='margin:10px;padding:10px' class='table col-md-6'> | + | <table class='table col-md-6 table-bordered'> |
− | <tr> | + | <tr style="color:#606060"> |
| <td>Spacer Length</td> | | <td>Spacer Length</td> |
| <td>19</td><td>20</td><td>21</td> | | <td>19</td><td>20</td><td>21</td> |
| <td>22</td><td>23</td><td>19-23</td> | | <td>22</td><td>23</td><td>19-23</td> |
| </tr> | | </tr> |
− | <tr> | + | <tr style="color:#606060"> |
| <td>Marker Number</td> | | <td>Marker Number</td> |
| <td>690</td><td>836</td><td>746</td> | | <td>690</td><td>836</td><td>746</td> |
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| </tr> | | </tr> |
| </table> | | </table> |
− | <p class='col-md-12'>The number of gRNA pairs under the condition of different spacer length between left and right pair from 19 to 23 bps in Mycobacterium Tuberculosis genome</p> | + | <p class='col-md-12'>The number of gRNA pairs under the condition of different spacer length between left and right pair from 19 to 23 bps in <i>Mycobacterium Tuberculosis</i> genome</p> |
| </div> | | </div> |
| </div> | | </div> |
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| <div class='col-md-2'></div> | | <div class='col-md-2'></div> |
| </div> | | </div> |
| + | <div id="Modeling-OligoGenerator"> |
| + | <p> </p> |
| + | </div> |
| <div class="col-md-12"> | | <div class="col-md-12"> |
− | <p><b>Figure 6.</b> 72 target sites (MTB-specific markers) out of 9 fragments on MTB genome, screened out using SSPD.)</p> | + | <p ><b>Figure 6.</b> 72 target sites (MTB-specific markers) out of 9 fragments on MTB genome, screened out using SSPD.)</p> |
| </div> | | </div> |
| </div> | | </div> |
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| <!--End of Modeling-SSPD--> | | <!--End of Modeling-SSPD--> |
| <!--Start Oligo Generator--> | | <!--Start Oligo Generator--> |
− | <div id="Modeling-OligoGenerator" class="col-md-12" style="padding:0"> | + | <div class="col-md-12" style="padding:0"> |
| <h3 class="classic-title" style='margin-top:50px'><span>Oligo Generator</span></h3> | | <h3 class="classic-title" style='margin-top:50px'><span>Oligo Generator</span></h3> |
− | <p>Using SSPD method mentioned above, we can easily find the reliable target sites on genome. However, designing multiple target sites into oligonucleotides sequences for following sgRNA construction manually can be laborious. Thus here we developed a supplementary program to facilitate oligo sequence generation, which is combined with our sgRNA generator (Link to Part xxx). Specifically, we used Golden Gate Cloning to make it more convenient to substitute guide sequences for different target sites. Detailed operation is explained on flow chart below. See more details in <a href="" style="color:#00afd1">Supplementary Information 2</a>. | + | <p>Using SSPD method mentioned above, we can easily find the reliable target sites on genome. However, designing multiple target sites into oligonucleotides sequences for following sgRNA construction manually can be laborious. Thus here we developed a supplementary program to facilitate oligo sequence generation, which is combined with our sgRNA generator (Link to Part xxx). Specifically, we used Golden Gate Cloning to make it more convenient to substitute guide sequences for different target sites. Detailed operation is explained on flow chart below. See more details in <a href="https://static.igem.org/mediawiki/2015/a/a7/Peking-Modeling-Supplementary.pdf" style="color:#00afd1">Supplementary Information 2</a>. |
| </p> | | </p> |
| <div id="Modeling_Fig7"> | | <div id="Modeling_Fig7"> |
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| <!-- Start Subscribe & Social Links Widget --> | | <!-- Start Subscribe & Social Links Widget --> |
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| <div class="col-md-12 footer-widget social-widget row" style="padding-top:0:padding-bottom:0;margin-top:0;margin-bottom:0"> | | <div class="col-md-12 footer-widget social-widget row" style="padding-top:0:padding-bottom:0;margin-top:0;margin-bottom:0"> |
| <h4>Share This<span class="head-line"></span></h4> | | <h4>Share This<span class="head-line"></span></h4> |
| <ul class="social-icons" style="padding-top:0:padding-bottom:0;margin-top:0;margin-bottom:0"> | | <ul class="social-icons" style="padding-top:0:padding-bottom:0;margin-top:0;margin-bottom:0"> |
| <li style="padding-top:0:padding-bottom:0;margin-top:0;margin-bottom:0"><!--HERE need to be changed--> | | <li style="padding-top:0:padding-bottom:0;margin-top:0;margin-bottom:0"><!--HERE need to be changed--> |
− | <a class="twitter" target="_blank" href="https://twitter.com/home/?status=https://2015.igem.org/Team:Peking/Practices/Background"> | + | <a class="twitter" target="_blank" href="https://twitter.com/home/?status=https://2015.igem.org/Team:Peking"> |
| <i class="fa"> | | <i class="fa"> |
| <img src="https://static.igem.org/mediawiki/2015/d/d0/Peking-twitter.png" style="height:20px;width:auto; margin-top:7px;"> | | <img src="https://static.igem.org/mediawiki/2015/d/d0/Peking-twitter.png" style="height:20px;width:auto; margin-top:7px;"> |
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| <li style="padding-top:0:padding-bottom:0;margin-top:0;margin-bottom:0"><!--HERE need to be changed--> | | <li style="padding-top:0:padding-bottom:0;margin-top:0;margin-bottom:0"><!--HERE need to be changed--> |
− | <a class="facebook"target="_blank" href="https://www.facebook.com/sharer/sharer.php?u=https://2015.igem.org/Team:Peking/Practices/Background"> | + | <a class="facebook"target="_blank" href="https://www.facebook.com/sharer/sharer.php?u=https://2015.igem.org/Team:Peking"> |
| <i class="fa"> | | <i class="fa"> |
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| <li style="padding-top:0:padding-bottom:0;margin-top:0;margin-bottom:0"><!--HERE need to be changed--> | | <li style="padding-top:0:padding-bottom:0;margin-top:0;margin-bottom:0"><!--HERE need to be changed--> |
− | <a class="google"target="_blank" href="https://plus.google.com/share?url=https://2015.igem.org/Team:Peking/Practices/Background"> | + | <a class="google"target="_blank" href="https://plus.google.com/share?url=https://2015.igem.org/Team:Peking"> |
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| <img src="https://static.igem.org/mediawiki/2015/7/7a/Peking-google_plus.png" style="height:20px; margin-top:7px;"> | | <img src="https://static.igem.org/mediawiki/2015/7/7a/Peking-google_plus.png" style="height:20px; margin-top:7px;"> |