Team:Aberdeen Scotland/Parts

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<li class="curr"><a class="curr" href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts">Background</a></li>
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<li class="curr"><a class="curr" href="#">Created</a></li>
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<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2000">Bba_K1352000</a></li>
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<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2001">Bba_K1352001</a></li>
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<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2002">Bba_K1352002</a></li>
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<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2003">Bba_K1352003</a></li>
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<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2004">Bba_K1352004</a></li>
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<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2006">Bba_K1352006</a></li>
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<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/Device">Device Data</a></li>
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<li class="curr"><a class="curr" href="#">Improved</a></li>
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<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_9001">Bba_K759001</a></li>
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<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2009">Bba_K542009</a></li>
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<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_6007">Bba_K346007</a></li>
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<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_0000">Bba_K1090000</a></li>
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<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_9002">Bba_T9002</a></li>
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<h1 >WELCOME TO 2014 Aberdeen Scotland iGEM page! </h1>
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<p>Our team page is currently under construction. Please check back again for more updates!
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<br>On this page we will document our project, introduce our team members, our progress <br> and share our iGEM experience with the rest of the world! </p>
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<div class="t_overview">
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<h1><br>Background to Parts Design</h1>
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<p><b>Antigen 43</b> (sometimes called Ag43 or fluffing protein) is a phase-variable outer membrane protein encoded by flu gene. It is native to E.Coli K12 strain
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and is usually expressed at about 50, 000 copies/cell. Ag34 precursor is 1039 amino acids long and subsequently becomes cleaved into alpha and beta chains (499 and
 +
488 amino acids long respectively). The beta subunit forms a β-barrel pore via which alpha-subunit translocates to the cell surface, and with which it remains non-
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covalently joined. The surface alpha chain can be released by a brief heat treatment at approx. 60<sup>o</sup>C.</p>
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<p>Ag43 is an autotransporter protein, therefore it possesses all information necessary for translocation to the cell surface in its coding sequence.</p>
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<p>Ag43 mediates autoaggregation, via a velcro-like mechanism (Heras et. al., 2014), and plays a role in <i>E.coli</i> biofilm formation.</p>
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<p>Interestingly, the alpha subunit is able to express foreign peptide sequences on E.coli cell surface if inserted just in front of codon 148 (Kjærgaard et. al.,
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2002).</p><br><center>
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<img src="https://static.igem.org/mediawiki/2014/f/f1/Ag43_back.png"><br>
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<span style="font-size:11px">Fig.1&nbsp;&nbsp;&nbsp;&nbsp;Graphic representation of Ag43 autotransporter structure and process of autotransportation. <br>Source: Kjærgaard et. al., 2000; Van der Woude & Henderson, 2008 (modified).</span></p></center><br>
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<p>The shape of α-subunit of Ag43 resembles letter L (Fig.2). It consists of a 'β-helix domain [which forms] the stem of the letter L, followed by three rungs
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flanked by four β-hairpin motifs that bend the protein by about 110° and a C-terminal (...) parallel β-helix domain [which forms] the bottom of the letter L' (Heras
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et. al., 2014). It has been indicated that this unique shape plays a crucial role in cell-to-cell aggregation via velcro-like mechanism, in which α-subunits form a
 +
dimer by coling around each other. This interaction is strengthened by Van der Waals interactions, hydrogen bonds and salt bridges facilitated by the L-shape (Heras
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et. al., 2014). Recent research demonstrates that disruption of the bend and straightening of the shape by removal of two β-hairpin sequences eliminates self-
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association of Ag43 proteins (Heras et. al., 2014). Removal of β-hairpins does not interfere with protein translocation to the cell surface membrane.</p>
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<center> <p>Hairpin 1 sequence&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;268&nbsp;&nbsp;&nbsp;AATVTGTNRLGAFSVVA&nbsp;&nbsp;&nbsp;284</p>
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<p>Hairpin 2 sequence&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;341&nbsp;&nbsp;&nbsp;GAAVSGTRSDGKAFSIG&nbsp;&nbsp;&nbsp;357</p><br></center>
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<center><img src="https://static.igem.org/mediawiki/2014/b/b8/Ag43_small.png"><br>
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<p><span style="font-size:11px">Fig.2&nbsp;&nbsp;&nbsp;A) Graphic representation of Ag43 alpha-subunit; B) Interaction between two alpha-subunits in a velcro-like mechanism of auto
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-aggregation; C) Disruption of L-shape and linearization of the alpha-subunit eliminates auto-aggregation properties.</span></p></center><br><br>
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<p><b>Ice Nucleation Protein (INP)</b> is used by bacteria in nature to nucleate ice crystals at slightly sub-zero temperatures; these crystals cause frost-damage to
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plant tissues which releases nutrients allowing the bacteria to metabolise them.  It is another autotransporter and is extremely similar to Ag43.  It also has an
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alpha and beta region and inserts itself into the cell’s outer membrane in basically the same way indicated in the first diagram above.  Unlike Ag43 where the
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foreign proteins are inserted at codon 148, in INP the protein is inserted on the C terminus.  INP has been used by a number of researchers and iGEM teams to surface-
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display proteins of interest. Ag43 is perhaps a better surface-displayer as it protrudes further from the cell surface than INP, but INP is easier to engineer as it
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has a larger carrying-capacity and the gene is much smaller.</p>
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<td > <h3>What information do I need to start putting my parts on the Registry? </h3></td>
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An important aspect of the iGEM competition is the use and creation of standard  biological parts. Each team will make new parts during iGEM and will submit them to the <a href="http://partsregistry.org"> Registry of Standard Biological Parts</a>. The iGEM software provides an easy way to present the parts your team has created. The "groupparts" tag will generate a table with all of the parts that your team adds to your team sandbox. 
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<p>
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<strong>Note that if you want to document a part you need to document it on the <a href="http://partsregistry.org Registry"> Registry</a>, not on your team wiki.</strong> Future teams and other users and are much more likely to find parts on the Registry than on your team wiki.
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Remember that the goal of proper part documentation is to describe and define a part, so that it can be used without a need to refer to the primary literature. Registry users in future years should be able to read your documentation and be able to use the part successfully. Also, you should provide proper references to acknowledge previous authors and to provide for users who wish to know more.
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<h3>When should you put parts into the Registry?</h3>
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As soon as possible! We encourage teams to start completing documentation for their parts on the Registry as soon as you have it available. The sooner you put up your parts, the better recall you will have of all details surrounding your parts. Remember you don't need to send us the DNA to create an entry for a part on the Registry. However, you must send us the sample/DNA before the Jamboree. Only parts for which you have sent us samples/DNA are eligible for awards and medal requirements.
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The information needed to initially create a part on the Registry is:
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<li>Part Name</li>
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<li>Sequence</li>
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<li>Short Description (60 characters on what the DNA does)</li>
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<li>Long Description (Longer description of what the DNA does)</li>
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<li>Design considerations</li>
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We encourage you to put up <em>much more</em> information as you gather it over the summer. If you have images, plots, characterization data and other information, please also put it up on the part page. Check out part <a href="http://parts.igem.org/Part:BBa_K404003">BBa_K404003</a> for an excellent example of a highly characterized part.
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You can add parts to the Registry at our <a href="http://parts.igem.org/Add_a_Part_to_the_Registry"> Add a Part to the Registry</a> link.
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<tr><td colspan="3" > <h3> Parts Table</h3></td></tr>
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Any parts your team has created will appear in this table below:</td></tr>
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<p style="color:#E7E7E7" align="right"> <a href="https://2014.igem.org/wiki/index.php?title=Team:Aberdeen_Scotland/Parts&action=edit"style="color:#FF0000"> Edit</a> </p>
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<groupparts>iGEM013 Aberdeen_Scotland</groupparts>
 

Latest revision as of 02:09, 18 October 2014

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




Background to Parts Design


Antigen 43 (sometimes called Ag43 or fluffing protein) is a phase-variable outer membrane protein encoded by flu gene. It is native to E.Coli K12 strain and is usually expressed at about 50, 000 copies/cell. Ag34 precursor is 1039 amino acids long and subsequently becomes cleaved into alpha and beta chains (499 and 488 amino acids long respectively). The beta subunit forms a β-barrel pore via which alpha-subunit translocates to the cell surface, and with which it remains non- covalently joined. The surface alpha chain can be released by a brief heat treatment at approx. 60oC.

Ag43 is an autotransporter protein, therefore it possesses all information necessary for translocation to the cell surface in its coding sequence.

Ag43 mediates autoaggregation, via a velcro-like mechanism (Heras et. al., 2014), and plays a role in E.coli biofilm formation.

Interestingly, the alpha subunit is able to express foreign peptide sequences on E.coli cell surface if inserted just in front of codon 148 (Kjærgaard et. al., 2002).



Fig.1    Graphic representation of Ag43 autotransporter structure and process of autotransportation.
Source: Kjærgaard et. al., 2000; Van der Woude & Henderson, 2008 (modified).


The shape of α-subunit of Ag43 resembles letter L (Fig.2). It consists of a 'β-helix domain [which forms] the stem of the letter L, followed by three rungs flanked by four β-hairpin motifs that bend the protein by about 110° and a C-terminal (...) parallel β-helix domain [which forms] the bottom of the letter L' (Heras et. al., 2014). It has been indicated that this unique shape plays a crucial role in cell-to-cell aggregation via velcro-like mechanism, in which α-subunits form a dimer by coling around each other. This interaction is strengthened by Van der Waals interactions, hydrogen bonds and salt bridges facilitated by the L-shape (Heras et. al., 2014). Recent research demonstrates that disruption of the bend and straightening of the shape by removal of two β-hairpin sequences eliminates self- association of Ag43 proteins (Heras et. al., 2014). Removal of β-hairpins does not interfere with protein translocation to the cell surface membrane.

Hairpin 1 sequence      268   AATVTGTNRLGAFSVVA   284

Hairpin 2 sequence      341   GAAVSGTRSDGKAFSIG   357



Fig.2   A) Graphic representation of Ag43 alpha-subunit; B) Interaction between two alpha-subunits in a velcro-like mechanism of auto -aggregation; C) Disruption of L-shape and linearization of the alpha-subunit eliminates auto-aggregation properties.



Ice Nucleation Protein (INP) is used by bacteria in nature to nucleate ice crystals at slightly sub-zero temperatures; these crystals cause frost-damage to plant tissues which releases nutrients allowing the bacteria to metabolise them. It is another autotransporter and is extremely similar to Ag43. It also has an alpha and beta region and inserts itself into the cell’s outer membrane in basically the same way indicated in the first diagram above. Unlike Ag43 where the foreign proteins are inserted at codon 148, in INP the protein is inserted on the C terminus. INP has been used by a number of researchers and iGEM teams to surface- display proteins of interest. Ag43 is perhaps a better surface-displayer as it protrudes further from the cell surface than INP, but INP is easier to engineer as it has a larger carrying-capacity and the gene is much smaller.