Team:Alberta/ByteCreation

From 2009.igem.org

(Difference between revisions)
 
(27 intermediate revisions not shown)
Line 4: Line 4:
<style type="text/css">
<style type="text/css">
.b1f, .b2f, .b3f, .b4f{font-size:1px; overflow:hidden; display:block;}
.b1f, .b2f, .b3f, .b4f{font-size:1px; overflow:hidden; display:block;}
-
.b1f {height:1px; background:#ADED7C; margin:0 5px;}
+
.b1f {height:1px; background:#e1e1e1; margin:0 5px;}
-
.b2f {height:1px; background:#ADED7C; margin:0 3px;}
+
.b2f {height:1px; background:#e1e1e1; margin:0 3px;}
-
.b3f {height:1px; background:#ADED7C; margin:0 2px;}
+
.b3f {height:1px; background:#e1e1e1; margin:0 2px;}
-
.b4f {height:2px; background:#ADED7C; margin:0 1px;}
+
.b4f {height:2px; background:#e1e1e1; margin:0 1px;}
-
.content {background: #ADED7C;}
+
.content {background: #e1e1e1;}
.content div {margin-left: 5px;}
.content div {margin-left: 5px;}
</style>
</style>
Line 26: Line 26:
     <div class="Outreach">
     <div class="Outreach">
     <div style="height: 400; background:#FFFFFF; colorou line-height:100% padding: 3px 0px;">
     <div style="height: 400; background:#FFFFFF; colorou line-height:100% padding: 3px 0px;">
-
    <h1>BioBytes Plasmid Construction</h1>
+
 +
<h1>Byte Creation</h1>
 +
 +
    <h2>Standard Plasmids</h2>
<!-- <div align="justify" style="padding-left:20px; padding-right:20px"> -->
<!-- <div align="justify" style="padding-left:20px; padding-right:20px"> -->
<div align="justify">
<div align="justify">
-
<font size="2">
+
<p>Each part to be assembled via the BioBytes method needs to have its ends altered to either the AB or the BA Byte type. To do this, unique plasmids were developed and named <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K187000">pAB</a> and <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K187001">pBA</a> (<B>Figure 1</B>). These plasmids were designed and constructed from pUC19 and contain the pMB1 high copy origin. Unique cassettes were designed containing PstI, XbaI, and  primer annealing regions complementary to the ‘A’ and ‘B’ ends. The cassettes (<B>Figure 2</B>) were synthesized and inserted using two restriction sites (EcoRI and NsiI). This left an EcoRI site on the final plasmid as well a PstI scar site. The primer annealing regions in pAB are reverse to that in pBA so that compatible sticky ends can be produced in either plasmid. Genes can be inserted using XbaI and PstI (or an enzyme which produces a compatible sticky end). The plasmids were originally designed for a previous but now obsolete system, where the sticky ends were generated via nicking enzymes. </p>
-
<P>
+
-
<img src="https://static.igem.org/mediawiki/2009/a/ad/UofA09_splasmids_Test1.jpg">
+
<p><b>Figure 1.</b></p>
 +
<center>
 +
<table>
 +
<tr>
 +
<td>
 +
<img src="https://static.igem.org/mediawiki/2009/9/95/PAB.png" width="360">
 +
</td>
 +
<td>
 +
<img src="https://static.igem.org/mediawiki/2009/e/e7/PBA.png" width="360">
 +
</td>
 +
</tr>
 +
</table>
 +
</center>
 +
<p><b>Figure 2.</b></p>
 +
<center><img src="https://static.igem.org/mediawiki/2009/c/ce/UofA09_Bead_ABcassetteimg.png">
 +
<img src="https://static.igem.org/mediawiki/2009/9/96/UofA09_Bead_BAcassetteimg.png">
-
</P>
+
</center>
-
<P>
+
-
We believe that sharing one’s research with the community is an important responsibility, both to keep research accountable to the public and to open doors for the next generation to get involved. Even for those students who don’t pursue science, an exposure to what synthetic biology is allows them to make more informed, responsible choices as consumers and voters. Thus, through high school outreach, we’re setting a good example of good example of corporate social responsibility. </P>
+
-
<P>
+
-
Finally, we strive to learn how to better communicate synthetic biology to students. In order to evaluate the impact of our outreach, we collect feedback forms after presentations and have an online survey.
+
-
</p>
+
-
</font></div>
+
<h2>How To Create a Byte</h2>
 +
 +
 +
<p>In order to format a part as an AB or BA form Byte, the part first needs to be cloned in to pAB or pBA, respectively. This is done using a XbaI and PstI digest of both the insert and backbone, followed by ligation to place the part inside of the AB or BA cassette.</p>
-
      </div></div>
+
<p>Once the part is cloned, universal PCR primers containing deoxyuridine residues are used to amplify the part. To create an AB Byte from pAB, the universal primers <a href="http://partsregistry.org/wiki/index.php/Part:BBa_K187365">pAB_F</a> and <a href="http://partsregistry.org/wiki/index.php/Part:BBa_K187366">pAB_R</a> are used. For the creation of a BA Byte from pBA, the primers <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K187367">pBA_F</a> and <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K187368">pBA_R</a> are used (<B>Figure 3</B>).
-
<b class="b4f"></b><b class="b3f"></b><b class="b2f"></b><b class="b1f"></b>
+
-
    </td>
+
-
  </tr>
+
 +
<p><b>Figure 3.</b></p>
 +
<center>
 +
<img src="https://static.igem.org/mediawiki/2009/f/ff/UofA09_Bead_universalprimers.png">
 +
</center>
 +
After amplification, treatment with USER<sup>TM</sup> mix (available from New England Biolabs) creates a nucleotide gap at the position of the uracil by first excising the uracil base by Uracil DNA Glycosylase and then cleaving the phosphodiester backbone at the apyrimidinic site via Endonuclease VIII. The resulting short oligonucleotides are then purified away from the PCR product to produce mature 12 base 3' overhangs of the AB or BA form Byte (<B>Figure 4</B>).</p>
 +
 +
 +
<p><b>Figure 4.</b></p>
 +
<center>
 +
<img src="https://static.igem.org/mediawiki/2009/c/c3/Alberta_byteconstruction.png" width="500">
 +
</center>
 +
 +
<P>The protocol for amplifying and digesting AB and BA Bytes can be found in our <a href="https://2009.igem.org/Team:Alberta/Protocols">lab section</a>.
 +
 +
</P>
 +
</div>
-
<tr>
 
-
<td style="height: 400; padding-left: 10px; padding-right: 10px; padding-top: 11px;">
 
-
    <b class="b1f"></b><b class="b2f"></b><b class="b3f"></b><b class="b4f"></b>
 
-
    <div class="Outreach">
 
-
    <div style="height: 400; background:#FFFFFF; colorou line-height:100% padding: 3px 0px;">
 
-
    <h1>BioBytes Plasmid Construction</h1>
 
-
<!-- <div align="justify" style="padding-left:20px; padding-right:20px"> -->
 
-
<div align="justify">
 
-
<font size="2">
 
-
<P>
 
-
Our Human Practices component consists of presentations and pamphlets for high school and junior high school students about synthetic biology, iGEM, and our project. We’re doing this outreach primarily for the high school students’ own interest and benefit. Synthetic biology is rapidly changing the biotechnology industry, and an understanding of synthetic biology would enrich a student’s consideration of career choices. Becoming excited about a potential career option can also provide motivation for academic success. Our outreach is also a service to the research community, as the more students who know about synthetic biology, the more who may pursue it as a career. A greater pool of human resources can increase the quantity and quality of research </P>
 
-
<P>
 
-
We believe that sharing one’s research with the community is an important responsibility, both to keep research accountable to the public and to open doors for the next generation to get involved. Even for those students who don’t pursue science, an exposure to what synthetic biology is allows them to make more informed, responsible choices as consumers and voters. Thus, through high school outreach, we’re setting a good example of good example of corporate social responsibility. </P>
 
-
<P>
 
-
Finally, we strive to learn how to better communicate synthetic biology to students. In order to evaluate the impact of our outreach, we collect feedback forms after presentations and have an online survey.
 
-
</p>
 
-
</font></div>
 
       </div></div>
       </div></div>

Latest revision as of 03:29, 22 October 2009

University of Alberta - BioBytes










































































































Byte Creation

Standard Plasmids

Each part to be assembled via the BioBytes method needs to have its ends altered to either the AB or the BA Byte type. To do this, unique plasmids were developed and named pAB and pBA (Figure 1). These plasmids were designed and constructed from pUC19 and contain the pMB1 high copy origin. Unique cassettes were designed containing PstI, XbaI, and primer annealing regions complementary to the ‘A’ and ‘B’ ends. The cassettes (Figure 2) were synthesized and inserted using two restriction sites (EcoRI and NsiI). This left an EcoRI site on the final plasmid as well a PstI scar site. The primer annealing regions in pAB are reverse to that in pBA so that compatible sticky ends can be produced in either plasmid. Genes can be inserted using XbaI and PstI (or an enzyme which produces a compatible sticky end). The plasmids were originally designed for a previous but now obsolete system, where the sticky ends were generated via nicking enzymes.

Figure 1.

Figure 2.

How To Create a Byte

In order to format a part as an AB or BA form Byte, the part first needs to be cloned in to pAB or pBA, respectively. This is done using a XbaI and PstI digest of both the insert and backbone, followed by ligation to place the part inside of the AB or BA cassette.

Once the part is cloned, universal PCR primers containing deoxyuridine residues are used to amplify the part. To create an AB Byte from pAB, the universal primers pAB_F and pAB_R are used. For the creation of a BA Byte from pBA, the primers pBA_F and pBA_R are used (Figure 3).

Figure 3.

After amplification, treatment with USERTM mix (available from New England Biolabs) creates a nucleotide gap at the position of the uracil by first excising the uracil base by Uracil DNA Glycosylase and then cleaving the phosphodiester backbone at the apyrimidinic site via Endonuclease VIII. The resulting short oligonucleotides are then purified away from the PCR product to produce mature 12 base 3' overhangs of the AB or BA form Byte (Figure 4).

Figure 4.

The protocol for amplifying and digesting AB and BA Bytes can be found in our lab section.