Difference between revisions of "Team:Uppsala/Parts"

 
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              <h2 class="text"> Parts</h2>
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<h3 style="margin:auto; float:none;">CRISPR Biobricks</h3>
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During the summer we planned to build several biobricks. In first place we wanted to assemble a composite biobrick made of a pBAD/araC (BBa_I0500), a medium RBS (BBa_0032) our his-tagged CPF1 protein and a double terminator (BBa_0015). The arabinose induced promoter was chosen to avoid expression leakage since expression of endonucleases might lead to cell toxicity (Figure 1a).
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Secondly, we planned to build a pre-crRNA endowed of a double terminator (BBa_0015) that could be used to target any sequence the user wants to cleave (Figure 1b).
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<img class="img-responsive img-rounded" src="https://static.igem.org/mediawiki/2016/f/f8/T--Uppsala--CRISPR_Parts_fig4.png"/>
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<figcaption>Figure 1: Assembly of the CRISPR biobricks
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The spacer of this Biobrick was designed so that it could have been easily replaced with a simple digestion in order for future teams to design and synthesize only their 24 nucleotides long spacer sequence targeting the desired DNA, instead of the full CRISPR array. This pre-crRNA was designed in Full Length Repeat- Spacer- Full Length Repeat, where the repetitive regions were copied from F.tularensis subsp. Novicida 7 strain U112 (Genebank CP000439.1), and a random replaceable spacer sequence of 33 bp was inserted (33 base pairs were used because this was the length of the spacer introduced in between F.tularensis CPF1 CRISPR array. The spacer was endowed of the recognition sites for bsaI restriction enzymes. In this way, straggered cut with different overhangs occurs in the repetitive region, so that by designing the spacer with the correct overhangs, scarless insertion of any spacer into the array is possible (Figure 2).
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Designing of this sequence was inspired by the design used by Bikard et al. in 2013 for Cas9 CRISPR array plasmid. </p>
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<img class="img-responsive img-rounded" src="https://static.igem.org/mediawiki/2016/d/da/T--Uppsala--CRISPR_Parts_fig5.png"/>
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<figcaption>Figure 2: Schematic representation of the replaceable spacer. Digestion with BsaI allow insertion of the desired spacer
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<p>Each team will make new parts during iGEM and will submit them to the Registry of Standard Biological Parts. The iGEM software provides an easy way to present the parts your team has created. The <code>&lt;groupparts&gt;</code> tag (see below) will generate a table with all of the parts that your team adds to your team sandbox.</p>
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<h3 style="margin:auto; float:none;">UnaG Biobricks</h3>
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<p>Remember that the goal of proper part documentation is to describe and define a part, so that it can be used without needing 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.</p>
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As part of this year’s iGEM project, the newly discovered inducible green fluorescent protein UnaG was characterized. At a size of 15.6 kDa, it is about half the size of GFP. Hence, UnaG would be appropriate to use as a fusion protein. With this in mind, we designed three different UnaG BioBricks; UnaG with a 6xHis-tag (BBa_K2003010) for characterization of the protein, UnaG with a 6xHistag and a flexible linker (Gly-Ser-Gly)2 (BBa_K2003011), and lastly UnaG with only the flexible linker (BBa_K2003012).
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<h5>Note</h5>
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<p>Note that parts must be documented on the <a href="http://parts.igem.org/Main_Page"> Registry</a>. This page serves to <i>showcase</i> the parts you have made. Future teams and other users and are much more likely to find parts by looking in the Registry than by looking at your team wiki.</p>
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<h5>Adding parts to the registry</h5>
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<p>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.</p>
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<p>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 you will remember all the details about your parts. Remember, you don't need to send us the DNA sample before you create an entry for a part on the Registry. (However, you <b>do</b> need to send us the DNA sample before the Jamboree. If you don't send us a DNA sample of a part, that part will not be eligible for awards and medal criteria.)</p>
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<h5>What information do I need to start putting my parts on the Registry?</h5>
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<p>The information needed to initially create a part on the Registry is:</p>
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<li>Part Name</li>
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<li>Part type</li>
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<li>Creator</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. </p>
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The default UnaG BioBrick (BBa_K2003010) was designed and ordered from IDT with a hexahistidine tag on the 5’ end of the gene and a flexible linker (GSG)2 on the 3’ end. However, right upstream of the flexible linker, two stop codons were added, meaning that the flexible linker will not be translated from this gene. By PCR mutagenesis, these two stop codons were removed, resulting in BBa_K2003011 which contains both the flexible linker and the histidine tag. Lastly, PCR was performed in order to remove the 6xHis-tag from BBa_K2003011, resulting in a UnaG BioBrick with only the flexible linker.
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<img class="img-responsive img-rounded" src="https://static.igem.org/mediawiki/2016/3/31/T--Uppsala--UnaG_Biobrick.jpg"/>
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<figcaption>Figure 3: Schematic view of the three different UnaG BioBricks. From the top left is; BBa_K2003011, BBa_K2003012, and BBa_K2003010, respectively.
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                <p>Here are all the biobricks that we have submitted.
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<h5>Inspiration</h5>
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<groupparts>iGEM2016 Uppsala</groupparts>
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<p>We have a created  a <a href="http://parts.igem.org/Well_Documented_Parts">collection of well documented parts</a> that can help you get started.</p>
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<p> You can also take a look at how other teams have documented their parts in their wiki:</p>
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<li><a href="https://2014.igem.org/Team:MIT/Parts"> 2014 MIT </a></li>
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<li><a href="https://2014.igem.org/Team:Heidelberg/Parts"> 2014 Heidelberg</a></li>
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<li><a href="https://2014.igem.org/Team:Tokyo_Tech/Parts">2014 Tokyo Tech</a></li>
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<h5>Part Table </h5>
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<groupparts>iGEM2016 Example help</groupparts>
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Latest revision as of 13:41, 19 October 2016

Parts

CRISPR Biobricks

During the summer we planned to build several biobricks. In first place we wanted to assemble a composite biobrick made of a pBAD/araC (BBa_I0500), a medium RBS (BBa_0032) our his-tagged CPF1 protein and a double terminator (BBa_0015). The arabinose induced promoter was chosen to avoid expression leakage since expression of endonucleases might lead to cell toxicity (Figure 1a). Secondly, we planned to build a pre-crRNA endowed of a double terminator (BBa_0015) that could be used to target any sequence the user wants to cleave (Figure 1b).

Figure 1: Assembly of the CRISPR biobricks

The spacer of this Biobrick was designed so that it could have been easily replaced with a simple digestion in order for future teams to design and synthesize only their 24 nucleotides long spacer sequence targeting the desired DNA, instead of the full CRISPR array. This pre-crRNA was designed in Full Length Repeat- Spacer- Full Length Repeat, where the repetitive regions were copied from F.tularensis subsp. Novicida 7 strain U112 (Genebank CP000439.1), and a random replaceable spacer sequence of 33 bp was inserted (33 base pairs were used because this was the length of the spacer introduced in between F.tularensis CPF1 CRISPR array. The spacer was endowed of the recognition sites for bsaI restriction enzymes. In this way, straggered cut with different overhangs occurs in the repetitive region, so that by designing the spacer with the correct overhangs, scarless insertion of any spacer into the array is possible (Figure 2). Designing of this sequence was inspired by the design used by Bikard et al. in 2013 for Cas9 CRISPR array plasmid.

Figure 2: Schematic representation of the replaceable spacer. Digestion with BsaI allow insertion of the desired spacer

UnaG Biobricks

As part of this year’s iGEM project, the newly discovered inducible green fluorescent protein UnaG was characterized. At a size of 15.6 kDa, it is about half the size of GFP. Hence, UnaG would be appropriate to use as a fusion protein. With this in mind, we designed three different UnaG BioBricks; UnaG with a 6xHis-tag (BBa_K2003010) for characterization of the protein, UnaG with a 6xHistag and a flexible linker (Gly-Ser-Gly)2 (BBa_K2003011), and lastly UnaG with only the flexible linker (BBa_K2003012).

The default UnaG BioBrick (BBa_K2003010) was designed and ordered from IDT with a hexahistidine tag on the 5’ end of the gene and a flexible linker (GSG)2 on the 3’ end. However, right upstream of the flexible linker, two stop codons were added, meaning that the flexible linker will not be translated from this gene. By PCR mutagenesis, these two stop codons were removed, resulting in BBa_K2003011 which contains both the flexible linker and the histidine tag. Lastly, PCR was performed in order to remove the 6xHis-tag from BBa_K2003011, resulting in a UnaG BioBrick with only the flexible linker.

Figure 3: Schematic view of the three different UnaG BioBricks. From the top left is; BBa_K2003011, BBa_K2003012, and BBa_K2003010, respectively.

Here are all the biobricks that we have submitted.

<groupparts>iGEM2016 Uppsala</groupparts>