Team:CityU HK/notebook/lablog 3

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   <li><a href='https://2014.igem.org/Team:CityU_HK/notebook/lablog'><span>FadD & FadL Module</span></a></li>
   <li><a href='https://2014.igem.org/Team:CityU_HK/notebook/lablog'><span>FadD & FadL Module</span></a></li>
   <li><a href='https://2014.igem.org/Team:CityU_HK/notebook/lablog_2'><span>'TesA Module</span></a></li>
   <li><a href='https://2014.igem.org/Team:CityU_HK/notebook/lablog_2'><span>'TesA Module</span></a></li>
-
   <li class='last'><a href='#'><span>Desaturase Module</span></a></li>
+
   <li class='last'><a href='https://2014.igem.org/Team:CityU_HK/notebook/lablog_3'><span>Desaturase Module</span></a></li>
</ul>
</ul>
</div>
</div>
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                             <div class="st-content">
                             <div class="st-content">
                                 <h2 class="title"><b>Week 3 (13/7~19/7)</b></h2>
                                 <h2 class="title"><b>Week 3 (13/7~19/7)</b></h2>
-
                                 <p><b>-</b> Miniprep: extracted plasmids containing Δ9, Δ12 and Δ15 genes</p>
+
                                 <p><b>-</b> Plasmids containing theΔ9, Δ12 and Δ15 desaturase genes were extracted using commercial DNA extraction kits and digested either singly or doubly with the XbaI and PstI restriction enzymes.</p>
-
                                <p><b>-</b> Restriction cut at XbaI and PstI sites of each type of extracted plasmids</p>
+
                                 <h2 class="title"><b>Week 5 (27/7~31/7)</b></h2>
                                 <h2 class="title"><b>Week 5 (27/7~31/7)</b></h2>
-
                                 <p><b>-</b> Restriction cut at XbaI and SpeI for promoter R0010 from plasmid pSB1C3<br> ->no band with matching size of promoter showed after running gel</p>
+
                                 <p><b>-</b> Plasmid pSB1C3 (which carries the R0010 promoter) was doubly digested with the XbaI and SpeI restriction enzymes<br> -> However, there is no band matching the size of the promoter R0010</p>
-
                                 <p><b>-</b> Restriction cut at XbaI and PstI forΔ9, Δ12 andΔ15 genes</p>
+
                                 <p><b>-</b> Three plasmids that carry theΔ9, Δ12 andΔ15 desaturase genes were each doubly digested with XbaI and PstI</p>
-
                                <p><b>-</b> Restriction cut at XbaI and PstI on plasmid pSB1C3</p>
+
                                 <p><b>-</b> Plasmid pSB1C3 was restriction digested with XbaI and PstI</p>
-
                                 <p><b>-</b> Ligation of each gene into each pSB1C3 plasmid</p>
+
                             </div>  
                             </div>  
                         </li>
                         </li>
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                             <a href="#">August - Week 1 & 2(3/8~16/8)<span class="st-arrow">Open or Close</span></a>
                             <a href="#">August - Week 1 & 2(3/8~16/8)<span class="st-arrow">Open or Close</span></a>
                             <div class="st-content">
                             <div class="st-content">
-
                                 <p><b>-</b> Transformation of suspected-to-be modified plasmids into E.coli (DH5αK12 strain)</p>
+
                                 <p><b>-</b> The Δ9, Δ12 andΔ15 desaturase genes were individually subcloned into the XbaI / PstI site of pSB1C3 and transformed into E.coli DH5α (K12 strain)</p>
-
                                 <p><b>-</b> Check clone by colony PCR and gel electrophoresis<br> -> Three times of gel electrophoreses were carried<br> -> The results were not desired, either no band in gel photo or incorrect sizes of band</p>
+
                                 <p><b>-</b> Transformants were checked by colony PCR and gel electrophoresis<br> -> - Gel electrophoresis analyses were repeated 3 times<br> -> The results were not promising, either no band was observed in the gel or incorrect size bands were obtained</p>
-
                                 <p><b>-</b> Primer design for colony PCR & sequencing<br> -> The first designed primers were based on the original iGEM primers, i.e. VF2 and VR, but with extended annealing sequences. They were used for sequencing<br> -> The second designed primers included more annealing sequences and also RBS sequence. They would be used as the other approach if the processing transformed plasmids did not really have inserts</p>
+
                                 <p><b>-</b> Primer design for colony PCR and DNA sequencing<br> -> The primers were designed based on the original iGEM primers, i.e. VF2 and VR, with a few additional nucleotides. They were also used for DNA sequencing to verify the identity of the insert<br> -> The primers subsequently designed included additional nucleotide sequences and an RBS sequence.</p>
-
                                 <p><b>-</b> Extraction of plasmids containing RBS B0030 and Δ9, Δ12 andΔ15 genes with RBS sequences</p>
+
                                 <p><b>-</b> Plasmids containing the RBS B0030 and the Δ9, Δ12 andΔ15 genes were purified using commercial DNA extraction kits</p>
                             </div>   
                             </div>   
                         </li>
                         </li>
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                             <a href="#">August - Week 3 (17/8~23/8)<span class="st-arrow">Open or Close</span></a>
                             <a href="#">August - Week 3 (17/8~23/8)<span class="st-arrow">Open or Close</span></a>
                             <div class="st-content">
                             <div class="st-content">
-
                                 <p><b>-</b> Repeated restriction cut at XbaI and PstI sites of gene carrying plasmids</p>
+
                                 <p><b>-</b> The cloned Δ9, Δ12 andΔ15 genes in pSC1C3 were restriction cut with XbaI and PstI, and sequentially subcloned into the XbaI / PstI site of pSB1C3 and transformed into E. coli</p>
-
                                <p><b>-</b> ligation of genes to PSB1C3 with XbaI and PstI sites cut and transformation with newly extracted genes</p>
+
                                 <p><b>-</b> Transformants were checked by colony PCR with gene-specific primers targeting the Δ9, Δ12 orΔ15 desaturase genes<br> -> The transformed bacteria seemed to have the target inserts<br> -> PCR-positive colonies were confirmed by DNA sequencing to carry the desired gene inserts</p>
-
                                 <p><b>-</b> Colonies picked for further clone check with three types of primers, including the original iGEM primers and two newly designed primers<br> -> The transformed bacteria seemed to have target inserts after running gel</p>
+
                                 <p><b>-</b> Plasmids containing the Δ9, Δ12 andΔ15 genes (in the pSB1C3 backbone) were purified using commercial DNA extraction kits</p>
-
                                <p><b>-</b> Sequencing of the transformed bacteria<br> -> all sequences matched as expected</p>
+
                                 <p><b>-</b> The Δ9-containing plasmid was double digested with SpeI / PstI, and the Δ12- andΔ15- containing plasmids were double digested with XbaI / PstI </p>
-
                                 <p><b>-</b> Miniprep of the extracted plasmids with Δ9, Δ12 andΔ15 genes in pSB1C3 plasmids</p>
+
-
                                 <p><b>-</b> Restriction digestion at SpeI and PstI sites ofΔ9 gene containing plasmids, XbaI and PstU sites ofΔ12, Δ15 gene containing plasmids</p>
+
                             </div>   
                             </div>   
                         </li>
                         </li>
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                             <a href="#">August - Week 4 (24/8~30/8)<span class="st-arrow">Open or Close</span></a>
                             <a href="#">August - Week 4 (24/8~30/8)<span class="st-arrow">Open or Close</span></a>
                             <div class="st-content">
                             <div class="st-content">
-
                                 <p><b>-</b> Gel electrophoresis in order to collectΔ9 gene containing plasmids, Δ12 and Δ15 genes</p>
+
                                 <p><b>-</b> Gel electrophoresis was carried out to prepare the Δ9, Δ12 and Δ15 gene fragments for sequential cloning into pSB1C3 to obtain the Δ9-Δ12-Δ15 gene cluster in the pSB1C3 plasmid backbone</p>
-
                                 <p><b>-</b> Gel purification of the collected products</p>
+
                                 <p><b>-</b> Gel purification of the DNA fragments</p>
-
                                 <p><b>-</b> Ligation of Δ9 gene containing plasmids andΔ12 gene insert</p>
+
                                 <p><b>-</b> Ligation of Δ9 gene containing plasmids and the Δ12 and Δ15 gene inserts</p>
-
                                 <p><b>-</b> Transformation of suspected-to-be modified plasmids into E.coli (DH5αK12 strain)</p>
+
                                 <p><b>-</b> Transformation of ligation mixture into E.coli DH5α</p>
-
                                 <p><b>-</b> Clone check by PCR and gel electrophoresis to see if grown bacteria contained Δ9 and Δ12 genes or not<br> -> Result matched, transformation was sucessful</p>
+
                                 <p><b>-</b> Transformant colonies were checked by colony PCR and gel electrophoresis to determine if bacterial transformants contain the Δ9, Δ12 andΔ15 genes<br> -> Results confirmed that the transformation was successful</p>
                             </div>   
                             </div>   
                         </li>                             
                         </li>                             
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       <a href="http://www.cityu.edu.hk/" target="_blank"><img src="https://static.igem.org/mediawiki/2014/9/9c/CityU_HK_citylogo.png" width="95"></a>
       <a href="http://www.cityu.edu.hk/" target="_blank"><img src="https://static.igem.org/mediawiki/2014/9/9c/CityU_HK_citylogo.png" width="95"></a>
     <a href="http://www6.cityu.edu.hk/bhdbapp/deptweb/index.html" target="_blank"><img src="https://static.igem.org/mediawiki/2014/9/98/CityU_HK_bchlogo.png" width="80"></a>
     <a href="http://www6.cityu.edu.hk/bhdbapp/deptweb/index.html" target="_blank"><img src="https://static.igem.org/mediawiki/2014/9/98/CityU_HK_bchlogo.png" width="80"></a>
-
    <img src="https://static.igem.org/mediawiki/2014/a/aa/CityU_HK_Invitrogen_Logo.jpg" width="100">
+
      <img src="https://static.igem.org/mediawiki/2014/b/b1/CityU_HK_Invitrogen.png" width="100">
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     <img src="https://static.igem.org/mediawiki/2014/0/0a/CityU_HK_New_England_Biolabs_sponsor.png" width="135">
+
     <img src="https://static.igem.org/mediawiki/2014/7/7a/CityU_HK_Neblogocol.png" width="135">
-
     <img src="https://static.igem.org/mediawiki/2014/2/22/IDT-Logo.jpg" width="125">
+
     <img src="https://static.igem.org/mediawiki/2014/d/dc/CityU_HK_IDT-Logo.png" width="125">
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+
</div> <!-- end of col-md-7-->
</div> <!-- end of col-md-7-->
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<div class="col-md-5">
<div class="col-md-5">
       <h3>Stay connected!</h3>
       <h3>Stay connected!</h3>
       <p>email: <a href="#">cityuhk.igem@gmail.com</a></p>
       <p>email: <a href="#">cityuhk.igem@gmail.com</a></p>
-
       <a href="#"><img src="https://s3.amazonaws.com/sbweb/images/icon-facebook-grey-150x150.png" width="50" height="48"></a>
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       <a href="http://www.facebook.com/igem.2014CityU"><img src="https://static.igem.org/mediawiki/2014/3/31/CityU_HK_fb_logo.png" width="50" height="48"></a>
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       <a href="#"><img src="http://ibo2014.org/wordpress/wp-content/themes/ibo2014/img/navbar/twitter_icon.png" width="49" height="51"></a>
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       <a href="http://twitter.com/CityUHK_iGEM"><img src="http://ibo2014.org/wordpress/wp-content/themes/ibo2014/img/navbar/twitter_icon.png" width="49" height="51"></a>
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       <a href="#"><img src="http://3.bp.blogspot.com/-zNUyQIVgA8Q/Ue8USlgr8II/AAAAAAAAl1w/6ZCWqhS59Ts/s1600/youtube.png" width="52" height="50"></a>
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       <a href="#"><img src="https://static.igem.org/mediawiki/2014/1/12/CityU_HK_youtube_logo.png" width="52" height="50"></a>
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Latest revision as of 10:59, 16 October 2014

Bootstrap 101 Template

Desaturase Module's Lablog

  • Late JulyOpen or Close

    Week 3 (13/7~19/7)

    - Plasmids containing theΔ9, Δ12 and Δ15 desaturase genes were extracted using commercial DNA extraction kits and digested either singly or doubly with the XbaI and PstI restriction enzymes.

    Week 5 (27/7~31/7)

    - Plasmid pSB1C3 (which carries the R0010 promoter) was doubly digested with the XbaI and SpeI restriction enzymes
    -> However, there is no band matching the size of the promoter R0010

    - Three plasmids that carry theΔ9, Δ12 andΔ15 desaturase genes were each doubly digested with XbaI and PstI

    - Plasmid pSB1C3 was restriction digested with XbaI and PstI

  • August - Week 1 & 2(3/8~16/8)Open or Close

    - The Δ9, Δ12 andΔ15 desaturase genes were individually subcloned into the XbaI / PstI site of pSB1C3 and transformed into E.coli DH5α (K12 strain)

    - Transformants were checked by colony PCR and gel electrophoresis
    -> - Gel electrophoresis analyses were repeated 3 times
    -> The results were not promising, either no band was observed in the gel or incorrect size bands were obtained

    - Primer design for colony PCR and DNA sequencing
    -> The primers were designed based on the original iGEM primers, i.e. VF2 and VR, with a few additional nucleotides. They were also used for DNA sequencing to verify the identity of the insert
    -> The primers subsequently designed included additional nucleotide sequences and an RBS sequence.

    - Plasmids containing the RBS B0030 and the Δ9, Δ12 andΔ15 genes were purified using commercial DNA extraction kits

  • August - Week 3 (17/8~23/8)Open or Close

    - The cloned Δ9, Δ12 andΔ15 genes in pSC1C3 were restriction cut with XbaI and PstI, and sequentially subcloned into the XbaI / PstI site of pSB1C3 and transformed into E. coli

    - Transformants were checked by colony PCR with gene-specific primers targeting the Δ9, Δ12 orΔ15 desaturase genes
    -> The transformed bacteria seemed to have the target inserts
    -> PCR-positive colonies were confirmed by DNA sequencing to carry the desired gene inserts

    - Plasmids containing the Δ9, Δ12 andΔ15 genes (in the pSB1C3 backbone) were purified using commercial DNA extraction kits

    - The Δ9-containing plasmid was double digested with SpeI / PstI, and the Δ12- andΔ15- containing plasmids were double digested with XbaI / PstI

  • August - Week 4 (24/8~30/8)Open or Close

    - Gel electrophoresis was carried out to prepare the Δ9, Δ12 and Δ15 gene fragments for sequential cloning into pSB1C3 to obtain the Δ9-Δ12-Δ15 gene cluster in the pSB1C3 plasmid backbone

    - Gel purification of the DNA fragments

    - Ligation of Δ9 gene containing plasmids and the Δ12 and Δ15 gene inserts

    - Transformation of ligation mixture into E.coli DH5α

    - Transformant colonies were checked by colony PCR and gel electrophoresis to determine if bacterial transformants contain the Δ9, Δ12 andΔ15 genes
    -> Results confirmed that the transformation was successful

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