Team:Aberdeen Scotland/Parts/ 6007

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<li><a href="http://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2001">Bba_K1352001</a></li>
<li><a href="http://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2001">Bba_K1352001</a></li>
<li><a href="http://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2002">Bba_K1352002</a></li>
<li><a href="http://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2002">Bba_K1352002</a></li>
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<li><a href="http://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2003">Bba_K1352003</a></li>
<li><a href="http://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2004">Bba_K1352004</a></li>
<li><a href="http://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2004">Bba_K1352004</a></li>
<li><a href="http://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2006">Bba_K1352006</a></li>
<li><a href="http://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2006">Bba_K1352006</a></li>
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<div class="t_overview">
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<div class="t_overview"><br>
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<h1>Background to Parts Design</h1>
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<h1>Our characterisation of existing BioBrick BBa_K346007</h1>
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<h2>Antigen 43</h2>
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<h3>Aim</h3>
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To verify Assembly Standard 10 compliance<br><br>
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<h3>Method</h3>
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<u>Standard restriction digest and gel electrophoresis</u><br>
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Escherichia coli bacterial culture containing plasmids BBa_K346007 (coding region of Ag43 only), was inoculated overnight in LB-chloramphenicol medium in a shaking water bath at 37370C. Plasmid mini preps were conducted with a Qiagen plasmid mini prep kit. Single and double restriction digests were carried out accordingly:<br>
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<img src="http://parts.igem.org/wiki/images/d/dd/Restriction_digest_table.png">
<img src="http://parts.igem.org/wiki/images/d/dd/Restriction_digest_table.png">
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Restriction digests with EcoRI, XbaI, SpeI and PstI was performed to linearize the vector and to confirm the existence of unique restriction sites, as required by the iGEM biobrick Assembly standard 10 rules. Double restriction digests with EcoRI plus PstI and XbaI plus SpeI were performed to separate the BBa_K346007 insert from the pSB1C3 plasmid backbone. Digests were incubated at 37370C for 2 hours and analysed by gel electrophoresis. Theoretical maps were used to calculate the expected sizes of the fragments and to compare them against the gel electrophoresis results.<br><br>
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<h3>Results</h3>
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<img src="http://parts.igem.org/wiki/images/0/06/Restriction_digest_on_BBa_K346007.png">
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<img src="http://parts.igem.org/wiki/images/0/06/Restriction_digest_on_BBa_K346007.png"><br>
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<br>
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<font size="2">Fig.1&nbsp;&nbsp;&nbsp;&nbsp;Gel electrophoresis on standard restriction digests of BBa_K346007
<font size="2">Fig.1&nbsp;&nbsp;&nbsp;&nbsp;Gel electrophoresis on standard restriction digests of BBa_K346007
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Restriction digests were separated on a 0.8% agarose gel. Lane 1; NEB 1 kb size standards ladder, Lane 2; uncut plasmid, Lanes 3-6; plasmid cut with either EcoRI, XbaI, SpeI or PstI respectively, Lane 7; plasmid cut with EcoRI+PstI, Lane 8; plasmid cut with XbaI+SpeI.
<br><br>
<br><br>
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<center>
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<img src="http://parts.igem.org/wiki/images/d/dd/Restriction_digest_table.jpg">
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<img src="http://parts.igem.org/wiki/images/d/dd/Restriction_digest_table.jpg"><br>
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<br>
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<font size="2">Fig.2&nbsp;&nbsp;&nbsp;&nbsp;Fragment sizes resulted from standard restriction digests on BBa_K346007.
<font size="2">Fig.2&nbsp;&nbsp;&nbsp;&nbsp;Fragment sizes resulted from standard restriction digests on BBa_K346007.
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<p>Antigen 43 (Ag43), the product of the </i>flu</i> gene, is a cell-surface autotransporter protein found in <i>Escherichia coli</i>. It is expressed at about 50,
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000 copies/cell and is initially synthesised as a precursor of 1039 amino acids. Upon removal of the signal peptide, the protein is transported to the cell surface
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and is composed of an α subunit (499 amino acids) at the N-terminus and a β subunit (488 amino acids) at the C-terminus. Ag43 is mainly known to induce cell-to-cell
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<h3>Results</h3>
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aggregation and be involved in biofilm formation. However, as the necessary information required for auto transportation resides in the protein itself, the main of
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The pSB1C3-BBa_K346007 construct was shown to contain 6 additional PstI sites, as found in the wild-type gene of Antigen 43. This part is the basic part of BBa_K759001 Biobrick, which we have also shown that contains the same PstI sites (see Experience section of BBa_K759001).<br>
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our project was to use it as a platform for displaying specific peptides on the surface of <i>E. coli</i>.</p>
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Thus we conclude that the BioBrick BBa_K346007 originally deposited with iGEM did not meet assembly standard 10, and the 6 native PstI sites present in Ag43 had not in fact been removed as stated in the original documentation.<br>
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<img src="http://2014.igem.org/wiki/images/2/2e/Ag43.jpg" alt="Ag43">
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Based on the results mentioned above, pSB1C3-BBa_K759001 has been chosen as the subject for removal of the PstI sites to ensure that an Ag43 BioBrick is created that meets Assembly Standard 10 requirements (see the RFC10 compatible version of this Biobrick created by 2014 iGEM Aberdeen team:BBa_K1352000)<br><br>
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Latest revision as of 02:11, 18 October 2014

Team:Aberdeen Scotland/Parts - 2014.ogem.org




Our characterisation of existing BioBrick BBa_K346007

Antigen 43


Aim

To verify Assembly Standard 10 compliance

Method

Standard restriction digest and gel electrophoresis
Escherichia coli bacterial culture containing plasmids BBa_K346007 (coding region of Ag43 only), was inoculated overnight in LB-chloramphenicol medium in a shaking water bath at 37370C. Plasmid mini preps were conducted with a Qiagen plasmid mini prep kit. Single and double restriction digests were carried out accordingly:

Restriction digests with EcoRI, XbaI, SpeI and PstI was performed to linearize the vector and to confirm the existence of unique restriction sites, as required by the iGEM biobrick Assembly standard 10 rules. Double restriction digests with EcoRI plus PstI and XbaI plus SpeI were performed to separate the BBa_K346007 insert from the pSB1C3 plasmid backbone. Digests were incubated at 37370C for 2 hours and analysed by gel electrophoresis. Theoretical maps were used to calculate the expected sizes of the fragments and to compare them against the gel electrophoresis results.

Results


Fig.1    Gel electrophoresis on standard restriction digests of BBa_K346007

Restriction digests were separated on a 0.8% agarose gel. Lane 1; NEB 1 kb size standards ladder, Lane 2; uncut plasmid, Lanes 3-6; plasmid cut with either EcoRI, XbaI, SpeI or PstI respectively, Lane 7; plasmid cut with EcoRI+PstI, Lane 8; plasmid cut with XbaI+SpeI.


Fig.2    Fragment sizes resulted from standard restriction digests on BBa_K346007.


Results

The pSB1C3-BBa_K346007 construct was shown to contain 6 additional PstI sites, as found in the wild-type gene of Antigen 43. This part is the basic part of BBa_K759001 Biobrick, which we have also shown that contains the same PstI sites (see Experience section of BBa_K759001).
Thus we conclude that the BioBrick BBa_K346007 originally deposited with iGEM did not meet assembly standard 10, and the 6 native PstI sites present in Ag43 had not in fact been removed as stated in the original documentation.
Based on the results mentioned above, pSB1C3-BBa_K759001 has been chosen as the subject for removal of the PstI sites to ensure that an Ag43 BioBrick is created that meets Assembly Standard 10 requirements (see the RFC10 compatible version of this Biobrick created by 2014 iGEM Aberdeen team:BBa_K1352000)