Difference between revisions of "Team:USP UNIFESP-Brazil/Experiments"

Line 83: Line 83:
  
  
 +
<ul id=menu-lateral style="absolute; width:20%">
  
−
<!-- https://static.igem.org/mediawiki/2016/8/83/T--USP_UNIFESP-Brazil--experimentslogo.jpeg -->
+
<br>
 +
 +
<li><a href="https://2016.igem.org/Team:USP_UNIFESP-Brazil/Description">Description</a></p>
 +
<p></p>
 +
<li><a href="#ass">Assembly of spider silk gene sequence: Getting ready for expression!</a></p>
 +
<p></p>
  
 +
<!--
 +
<li><a href="https://2016.igem.org/Team:USP_UNIFESP-Brazil/Proof">Proof of Concept</a></p>
 +
<p></p>
 +
<li><a href="#res">Results</a></p>
 +
<p></p>
 +
<li><a href="#not">Notebook</a></p>
 +
<p></p>
 +
<li><a href="#saf">Safety</a></p>
 +
<p></p>
 +
<li><a href="#mod">Modelling</a></p>
  
 +
</ul>
 +
-->
 +
</div>
  
−
 
+
<div class="row meio">
−
<div class="row meio">
+
  
 
<div class="small-10 columns small-offset-2 titulo-verde">
 
<div class="small-10 columns small-offset-2 titulo-verde">
Line 102: Line 120:
  
 
</div>
 
</div>
 +
<div class="row meio">
 +
 +
<div class="small-10 columns small-offset-2 titulo-verde">
 +
<div class="small-11 small-offset-1 columns"><a name="ass"></a>
 +
<h2> Assembly of spider silk gene sequence: Getting ready for expression!</h2>
 +
</div>
 +
</div>
 +
 +
<div class="small-10 columns small-offset-2">
 +
<div class="small-10 small-offset-1 columns">
 +
 +
<h4 class= black><b>Assembly by cut&paste</b></h4>
 +
 +
 +
<p class=black>As we had trouble with our USER-specific primers request and could not start with the USER method, we started an alternative way to polymerize the MaSp1 monomers.
 +
Based on the Nature protocol of Teulé et al(ref) we designed a cut and paste technique, to duplicate the MaSp1 sequence at each round. Telué and collaborators use a cloning vector with a restriction site in its resistance gene and two complementary but non regenerable restriction sites, one at each side of the insert, which is the sequence that is going to be duplicated. The vector is cut in two parallel separated digestions, at one side of the insert and at the resistance gene, and on the other side of the insert and also at the resistance gene. In both digestions, two fragments will be generated: one that contains the insert and a part of the resistance gene, and one that does not contain the insert. Ligating the insert-containing fragments of the two different digestions together results in the duplication of the insert in a vector with a complete resistance gene. </p>
 +
<p class=black>Doing iGEM in labs without an established molecular biology line was challenging, and although some features may seem small, they were surely fruits of hard work!</p>
 +
 +
<h4 class= black><b>Positive results</b></h4>
 +
 +
<ul>
 +
<!--- <li class=black> We were able to purify a <i>Pfu</i>X7 polymerase and standardize a working PCR protocol </li> --->
 +
 +
<li class=black style="font-size:20px" >We were able to design and clone in pSB1C3 a protein domain part (<a href= http://parts.igem.org/Part:BBa_K2136002>BBa_K2136002</a>) and a working gene expression cassete for microalgae (<a href=http://parts.igem.org/Part:BBa_K2136010>BBa_K2136010</a>) ! </li>
 +
 +
<!-- https://static.igem.org/mediawiki/2016/8/83/T--USP_UNIFESP-Brazil--experimentslogo.jpeg -->

Revision as of 14:10, 19 October 2016

AlgAranha Team USP_UNIFESP-Brazil

iGEM 2016




Experiments

Assembly of spider silk gene sequence: Getting ready for expression!

Assembly by cut&paste

As we had trouble with our USER-specific primers request and could not start with the USER method, we started an alternative way to polymerize the MaSp1 monomers. Based on the Nature protocol of Teulé et al(ref) we designed a cut and paste technique, to duplicate the MaSp1 sequence at each round. Telué and collaborators use a cloning vector with a restriction site in its resistance gene and two complementary but non regenerable restriction sites, one at each side of the insert, which is the sequence that is going to be duplicated. The vector is cut in two parallel separated digestions, at one side of the insert and at the resistance gene, and on the other side of the insert and also at the resistance gene. In both digestions, two fragments will be generated: one that contains the insert and a part of the resistance gene, and one that does not contain the insert. Ligating the insert-containing fragments of the two different digestions together results in the duplication of the insert in a vector with a complete resistance gene.

Doing iGEM in labs without an established molecular biology line was challenging, and although some features may seem small, they were surely fruits of hard work!

Positive results

  • We were able to design and clone in pSB1C3 a protein domain part (BBa_K2136002) and a working gene expression cassete for microalgae (BBa_K2136010) !