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<li><a href="https://2017.igem.org/Team:NU_Kazakhstan/Model">Model</a></li> | <li><a href="https://2017.igem.org/Team:NU_Kazakhstan/Model">Model</a></li> | ||
<li><a href="https://2017.igem.org/Team:NU_Kazakhstan/IoP">Improvement of Parts</a></li> | <li><a href="https://2017.igem.org/Team:NU_Kazakhstan/IoP">Improvement of Parts</a></li> | ||
+ | <li><a href="https://2017.igem.org/Team:NU_Kazakhstan/FP">Future Plans</a></li> | ||
<li><a href="https://2017.igem.org/Team:NU_Kazakhstan/References">References</a></li> | <li><a href="https://2017.igem.org/Team:NU_Kazakhstan/References">References</a></li> | ||
</ul> | </ul> | ||
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<ul class="fh5co-sub-menu"> | <ul class="fh5co-sub-menu"> | ||
<li><a href="https://2017.igem.org/Team:NU_Kazakhstan/Protocols">Protocols</a></li> | <li><a href="https://2017.igem.org/Team:NU_Kazakhstan/Protocols">Protocols</a></li> | ||
− | <li><a href="https://2017.igem.org/Team:NU_Kazakhstan/Timeline"> | + | <li><a href="https://2017.igem.org/Team:NU_Kazakhstan/Timeline">Calendar</a></li> |
</ul> | </ul> | ||
</li> | </li> | ||
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− | <div id="fh5co-hero" style="background-image: url( | + | <div id="fh5co-hero" style="background-image: url('https://image.ibb.co/hTzVcb/design_f.jpg');"> |
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<a href="#fh5co-main" class="smoothscroll fh5co-arrow to-animate hero-animate-4"><i class="ti-angle-down"></i></a> | <a href="#fh5co-main" class="smoothscroll fh5co-arrow to-animate hero-animate-4"><i class="ti-angle-down"></i></a> | ||
<!-- End fh5co-arrow --> | <!-- End fh5co-arrow --> | ||
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</p> | </p> | ||
− | <img src="https://static.igem.org/mediawiki/2017/6/6e/Pcr_dessign.jpeg" alt="sequences"> | + | <center><img src="https://static.igem.org/mediawiki/2017/6/6e/Pcr_dessign.jpeg" width="90%" height="90%" alt="sequences"></center> |
<br> | <br> | ||
− | Figure 1. Ordered sequences from IDT | + | <center>Figure 1. Ordered sequences from IDT</center> |
+ | <h3> List of designed primers </h3> | ||
+ | |||
+ | <p> | ||
+ | <li>Fwd AphVIII (Primer 1) CTGGATTGGATGTGCTGTTGACCG </li> | ||
+ | <li>Rev AphVIII (Primer 2) GACAGTGGCTAATGGCTGAAACGC </li> | ||
+ | <li>Fwd ChrR (Primer 3) CTGGCAGGTACGATGTATCCTCGG </li> | ||
+ | <li>Rev ChrR (Primer 4) TGTAGGGAACTGGGTACGCAATCC </li> | ||
+ | <li>Fwd Chromodulin (Primer 5) CGTCAGGACACTTGTTGACTTGGC </li> | ||
+ | <li>Rev Chromodulin (Primer 6) TTGACCTGGTACGAAGTAGCCTGC </li> | ||
+ | <li>Fwd Supernova (Primer 7) AGATCCTAGTTTGACCTGCGTGCC </li> | ||
+ | <li>Rev Supernova (Primer 8) ATTGCTGGGAATCCTACGAGTCCG </li> | ||
+ | </p> | ||
+ | <br> | ||
+ | <p>As it can be seen, these 4 transcriptional units have overlapping regions of the length of 49 bp each for Gibson Assembly (Figure 2). </p> | ||
+ | <br> | ||
+ | <center><img src="https://static.igem.org/mediawiki/2017/a/a8/Gipson.jpeg" width="90%" height="90%" alt="sequences"></center> | ||
+ | <br> | ||
+ | <center>Figure 2. Gibson Assembly in silico (SnapGene)</center><br> | ||
+ | <p> | ||
+ | Final construct looks like this (Figure 3):</p><br> | ||
+ | <center><img src="https://static.igem.org/mediawiki/2017/c/c6/Construgd.jpeg" width="90%" height="90%" alt="sequences"></center> | ||
+ | <br> | ||
+ | <center>Figure 3. Final construct</center> | ||
</div> | </div> | ||
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<br><br> | <br><br> | ||
<div class="content-box animate-box" id="rp"> | <div class="content-box animate-box" id="rp"> | ||
− | <h2> | + | <h2>Design of primers for insertion into pChlamy_4 vector</h2> |
− | + | <p> | |
+ | pChlamy_4 vector ordered from company ThermoFisher was used to check presence of our proteins in C. reinhardtii by Western blotting. This vector is designed in a such way, so that after restriction-ligation cloning into multiple cloning sites, it is fused with resistance gene (Zeocin) and also has V5 epitope with 6xHis tag. During translation target protein is self-cleaved from resistance gene by FMDV 2A peptide. | ||
− | + | In order to clone our coding sequence into the pChlamy_4 vector, transcriptional units were PCR amplified using specially primers that added EcoRI from 5’ end and XbaI+PstI from 3’ end. In addition to this, start codon and stop codon were removed, because coding sequence was fused to the resistance gene and addition of additional protein tags require absence of stop codon. After successful PCR, obtained amplicon could be digested with EcoRI+XbaI for further protein analysis (Western Blot/protein purification). In case if protein is desired to be expressed without any additional tags, EcoRI+PstI cloning can be used (Figure 4). | |
− | + | </p> | |
− | + | ||
− | + | ||
− | + | <center><img src="https://static.igem.org/mediawiki/2017/b/bd/Pchlamy4.jpeg" width="90%" height="90%" alt="sequences"></center> | |
+ | <br> | ||
+ | <center>Figure 4. Scheme for insertion into pChlamy_4 vector</center> | ||
+ | <br> | ||
+ | <p>Primers were carefully designed in such a way so that everything remained in frame (Figure 5):</p><br> | ||
− | < | + | <center><img src="https://static.igem.org/mediawiki/2017/5/5a/Mcs_pchlamy.jpeg" width="90%" height="90%" alt="sequence"></center> |
+ | <br> | ||
− | + | <center>Figure 5. Careful design of primers for insertion into multiple cloning sites of pChlamy_4 vector (example on SuperNova coding sequence). | |
− | + | </center> | |
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</div> | </div> | ||
+ | |||
<br><br> | <br><br> | ||
− | <div class="content-box animate-box" id=" | + | <div class="content-box animate-box" id="fl"> |
− | <h2> | + | <h2>Design of primers for CPEC (Circular Polymerase Extension Cloning) cloning of our parts into pSB1C3 plasmid</h2> |
− | + | <p>pSB1C3 linearized plasmid backbone was PCR amplified by using these two following primers:</p> | |
− | + | <br>Rwd pSB1C3, 39bp | |
− | + | <li>tactagtagcggccgctgcagtccggcaaaaaagggcaa</li> | |
− | + | Rev pSB1C3, 41bp | |
− | + | <li>tctagaagcggccgcgaattccagaaatcatccttagcgaa</li> | |
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− | + | <p>Each part was also PCR amplified in such a way so that it had flanking BioBrick Prefix and Suffix (Figure 6). | |
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<br> | <br> | ||
− | + | Following primers were used for each part (3 bp from 5’ end corresponding to pSB1C3 backbone, BioBrick prefix/suffix and 16-20 bp of overlapping nucleotides with target region at 3’ end of each primer):</p> | |
− | + | ||
<br> | <br> | ||
− | + | Fwd ChrR, 43bp | |
− | <img src="https:// | + | <li>ctggaattcgcggccgcttctagatgagcgagaagctgcaggt</li> |
+ | Rev ChrR, 43bp | ||
+ | <li>gactgcagcggccgctactagtattagatcttcacgcgctgaa</li> | ||
+ | Fwd SuperNova, 43bp | ||
+ | <li>ctggaattcgcggccgcttctagatgggtagcgaggtcggtcc</li> | ||
+ | Rev SuperNova, 42bp | ||
+ | <li>gactgcagcggccgctactagtattatcctcatcggacccga</li> | ||
+ | Fwd promoter, 44bp | ||
+ | <li>ctggaattcgcggccgcttctagagacggcggggagctcgctga</li> | ||
+ | Rev promoter, 42bp | ||
+ | <li>gactgcagcggccgctactagtctgcaaatggaaacggcgac</li> | ||
+ | |||
+ | Fwd Chromodulin, 39bp | ||
+ | <li>ctggaattcgcggccgcttctagatggaggaggaggagg</li> | ||
+ | Rev Chromodulin, 39bp | ||
+ | <li>gactgcagcggccgctactagtttaatcatcgccctcct</li> | ||
+ | Fwd terminator, 44bp | ||
+ | <li>ctggaattcgcggccgcttctagagctccgtgtaaatggaggcg</li> | ||
+ | Rev terminator, 42bp | ||
+ | <li>gactgcagcggccgctactagtgcttcaaatacgcccagccc</li> | ||
+ | |||
+ | <center><img src="https://static.igem.org/mediawiki/2017/1/10/Cpec_scheme.jpeg" width="90%" height="90%" alt="sequence"></center> | ||
<br> | <br> | ||
− | |||
− | |||
− | |||
− | |||
− | < | + | <center>Figure 6. Circular Polymerase Extension Cloning scheme |
− | + | </center> | |
− | + | ||
− | + | ||
− | + | ||
+ | <p>After PCR purification of two amplicons (part and backbone), CPEC reaction was run. Then CPEC product was transformed into E. coli and plasmid extraction was performed.</p> | ||
</div> | </div> | ||
+ | <br><br> | ||
+ | |||
</div> | </div> | ||
<div class="col-md-4 col-md-pull-8 left-sidebar" id="fh5co-sidebar"> | <div class="col-md-4 col-md-pull-8 left-sidebar" id="fh5co-sidebar"> | ||
<div class="sidebar-box animate-box"> | <div class="sidebar-box animate-box"> | ||
− | <h3 class="sidebar-heading"><span class="border"></span> | + | <h3 class="sidebar-heading"><span class="border"></span>Design</h3> |
<ul class="sidebar-links"> | <ul class="sidebar-links"> | ||
− | <li><a id="intr_sb" href="#"> | + | <li><a id="intr_sb" href="#">Whole construct assembly design</a></li><br> |
− | <li><a id="rp_sb" href="#"> | + | <li><a id="rp_sb" href="#">Design of primers for insertion into pChlamy_4 vector</a></li><br> |
− | + | <li><a id="fl_sb" href="#">Design of primers for CPEC (Circular Polymerase Extension Cloning) cloning of our parts into pSB1C3 plasmid</a></li> | |
− | <li><a id=" | + | |
− | + | ||
</ul> | </ul> | ||
</div> | </div> |
Latest revision as of 02:32, 2 November 2017
Whole construct assembly design
Initially 4 transcriptional units were ordered from IDT company using iGEM promotion. These 4 units were designed (additional flanking 24 bp sequences) in a such way so that it could be amplified using PCR and appropriate primers (Figure 1).
List of designed primers
As it can be seen, these 4 transcriptional units have overlapping regions of the length of 49 bp each for Gibson Assembly (Figure 2).
Final construct looks like this (Figure 3):
Design of primers for insertion into pChlamy_4 vector
pChlamy_4 vector ordered from company ThermoFisher was used to check presence of our proteins in C. reinhardtii by Western blotting. This vector is designed in a such way, so that after restriction-ligation cloning into multiple cloning sites, it is fused with resistance gene (Zeocin) and also has V5 epitope with 6xHis tag. During translation target protein is self-cleaved from resistance gene by FMDV 2A peptide. In order to clone our coding sequence into the pChlamy_4 vector, transcriptional units were PCR amplified using specially primers that added EcoRI from 5’ end and XbaI+PstI from 3’ end. In addition to this, start codon and stop codon were removed, because coding sequence was fused to the resistance gene and addition of additional protein tags require absence of stop codon. After successful PCR, obtained amplicon could be digested with EcoRI+XbaI for further protein analysis (Western Blot/protein purification). In case if protein is desired to be expressed without any additional tags, EcoRI+PstI cloning can be used (Figure 4).
Primers were carefully designed in such a way so that everything remained in frame (Figure 5):
Design of primers for CPEC (Circular Polymerase Extension Cloning) cloning of our parts into pSB1C3 plasmid
pSB1C3 linearized plasmid backbone was PCR amplified by using these two following primers:
Rwd pSB1C3, 39bp
Each part was also PCR amplified in such a way so that it had flanking BioBrick Prefix and Suffix (Figure 6).
Following primers were used for each part (3 bp from 5’ end corresponding to pSB1C3 backbone, BioBrick prefix/suffix and 16-20 bp of overlapping nucleotides with target region at 3’ end of each primer):
Fwd ChrR, 43bp
After PCR purification of two amplicons (part and backbone), CPEC reaction was run. Then CPEC product was transformed into E. coli and plasmid extraction was performed.