Difference between revisions of "Team:Rheda Bielefeld/Parts"

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<h1>Parts</h1>
 
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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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<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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<img src="https://static.igem.org/mediawiki/2018/1/11/T--Rheda_Bielefeld--parts%2Cquer.jpeg" width="50%">
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<h3>Note</h3>
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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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<h3>Adding parts to the registry</h3>
 
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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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<a href="http://parts.igem.org/Add_a_Part_to_the_Registry">
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<h2>  Plasmid Designs</h2>
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ADD PARTS
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</a>
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<a href="https://static.igem.org/mediawiki/2018/thumb/2/2b/T--Rheda_Bielefeld--cloning%2Csnapgene%28psb1C3_sub_pelB%29.jpeg/598px-T--Rheda_Bielefeld--cloning%2Csnapgene%28psb1C3_sub_pelB%29.jpeg.png">
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<img src="https://static.igem.org/mediawiki/2018/thumb/2/2b/T--Rheda_Bielefeld--cloning%2Csnapgene%28psb1C3_sub_pelB%29.jpeg/598px-T--Rheda_Bielefeld--cloning%2Csnapgene%28psb1C3_sub_pelB%29.jpeg.png" style="height:auto; width:100%;font-size:0.7em;"></a>Click the image to enlarge it</img>
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<br><br>
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Fig. 1: pelB-gene from the B.sub. in psb1C3-vector
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<br><br>
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<a href="https://static.igem.org/mediawiki/2018/thumb/8/88/T--Rheda_Bielefeld--cloning%2Csnapgene%28psb1C3_sub_yesZ%29.jpeg/582px-T--Rheda_Bielefeld--cloning%2Csnapgene%28psb1C3_sub_yesZ%29.jpeg.png">
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<img src="https://static.igem.org/mediawiki/2018/thumb/8/88/T--Rheda_Bielefeld--cloning%2Csnapgene%28psb1C3_sub_yesZ%29.jpeg/582px-T--Rheda_Bielefeld--cloning%2Csnapgene%28psb1C3_sub_yesZ%29.jpeg.png" style="height:auto; width:100%;font-size:0.7em;"></a>Click to enlarge</img>
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<br><br>
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Fig. 2: yesZ-gene from the B.sub. in psb1C3-vector
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<br><br>
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<a href="https://static.igem.org/mediawiki/2018/thumb/4/4c/T--Rheda_Bielefeld--cloning%2Csnapgene%28psb1C3_xan_pelB%29.jpeg/601px-T--Rheda_Bielefeld--cloning%2Csnapgene%28psb1C3_xan_pelB%29.jpeg.png">
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<img src="https://static.igem.org/mediawiki/2018/thumb/4/4c/T--Rheda_Bielefeld--cloning%2Csnapgene%28psb1C3_xan_pelB%29.jpeg/601px-T--Rheda_Bielefeld--cloning%2Csnapgene%28psb1C3_xan_pelB%29.jpeg.png" style="height:auto; width:100%;font-size:0.7em;"></a>Click to enlarge</img>
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<br><br>
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Fig. 3: pelB-gene from the Xan. in psb1C3-vector
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<h2> Parts</h2>
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The main idea of our project is to detect pollen by a DNA-based method, but another plan has been also to differ between the different types of pollen. Concerning this, we mainly focused on the pollen most commonly causing allergic reactions in our area. To analyze and to extract the DNA, we had to break open the outer layer of the pollen. Some attempts to mechanically or chemicaly open pollen are described here:
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<a href="https://2018.igem.org/Team:Rheda_Bielefeld/Pollen" style="color:yellow">Pollen</a><br>
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Pollen have an outer (exine) and inner (intine) layer, parts of which consist of pectine and cellulose <a href="https://2018.igem.org/Team:Rheda_Bielefeld/Assays" style="color:yellow"> (Assays)</a>. Therefore,we wanted to use Escherichia coli to produce enzymes which are able to dismantle these substances and hereby break open the pollen.
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<br>
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For this purpose we chose the following enzymes:
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<br>
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Out of the Bacillus subtilis the genes pelB and yesZ and out of Xanthomonas campestris the gene pelB. The genes pelB out of B.sub. and Xan. Code for pectinases or pectin lyases, yesZ being a beta-glucosidase, which could have enhanced the further dismanteling of the shell.
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<br>
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The BioBricks are supposed to be assembled performing a Gibson Assembly. For this we isolated the DNA of Bacillus subtilis and Xanthomonas campestris B100 and amplified the genes with specific primers. We wanted to clone the genes into psb1C3 and send them in. Using the E.coli vector pz9 availible to us in our lab we planned to characterize the genes. All in all we were trying to perform 6 clonings (3 clonings into psb1C3 and 3 into pz9). The psb1C3 constructs can be seen on the left. The experimentdesign was created with snapgene.
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We also thought about using other, already in iGEM existing, enzymes:
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<table style="width 100%">
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<th> Team </th>
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<th> Part </th>
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<th> Comments </th>
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<th> WLC Milwaukee <br> 2014 </th>
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<th> BBa_K1175007 <br> yesZ frim Bacillus Subtilis </th>
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<th> characterized in vitro assy <br> Statuts: It`s complicated </th>
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<th> Edinburgh 2008 </th>
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<th> BBa_K118023 <br> Codon optimized Caulobacter crescentus </th>
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<th> Caulobacter crescentus codon usage </th>
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<th> British Columbia 2016 </th>
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<th> BBa_K2139003 <br> Codon optimized Caulobacter crescentus </th>
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<th> sample not in stock <br> Endo-beta-1,4-glucanase E1 </th>
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<th> Stanford-Brown-Spelman 2014 </th>
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<th> BBa_K1499501 <br> Endo-1,4-&-beta-glucanase (EG1) / Neisseria sicca </th>
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<th> Not characterized, but Neisseria sicca is an S2 organism </th>
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The experiments with liquid nitrogen and trypsin as well as the primer design strategy were especially supported by the supervisors. No antibiotics were handled by team members.
  
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<h3>Inspiration</h3>
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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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<h2> What went wrong?</h2>
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Why didn’t the colonings work? Possible reasons for our failure are that we tried to perform a PCR using the wrong primers or that we committed an error somewhere else in the procedure. Another possibility is that we had the wrong stem of Bacillus subtilis from the beginning, because only the stem 168 has the required genes.
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We also tried to order the parts for our clonings from IDT, but they were not able to send them to us in time for us to still work with them.
  
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<h3>What information do I need to start putting my parts on the Registry?</h3>
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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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<h3>Part Table </h3>
 
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<p>Please include a table of all the parts your team has made during your project on this page. Remember part characterization and measurement data must go on your team part pages on the Registry. </p>
 
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<groupparts>iGEM18 Rheda_Bielefeld</groupparts>
 
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Latest revision as of 14:15, 17 October 2018

Home
The Team Our School Bielefeld University Rheda
Description Notebook
Overview Methods Pollen PCR Assays Cloning Strategy
Human Practices Marta Event
Safety Form Biosafety
Attributions Partners

Plasmid Designs

Click the image to enlarge it

Fig. 1: pelB-gene from the B.sub. in psb1C3-vector

Click to enlarge

Fig. 2: yesZ-gene from the B.sub. in psb1C3-vector

Click to enlarge

Fig. 3: pelB-gene from the Xan. in psb1C3-vector

Parts

The main idea of our project is to detect pollen by a DNA-based method, but another plan has been also to differ between the different types of pollen. Concerning this, we mainly focused on the pollen most commonly causing allergic reactions in our area. To analyze and to extract the DNA, we had to break open the outer layer of the pollen. Some attempts to mechanically or chemicaly open pollen are described here: Pollen
Pollen have an outer (exine) and inner (intine) layer, parts of which consist of pectine and cellulose (Assays). Therefore,we wanted to use Escherichia coli to produce enzymes which are able to dismantle these substances and hereby break open the pollen.
For this purpose we chose the following enzymes:
Out of the Bacillus subtilis the genes pelB and yesZ and out of Xanthomonas campestris the gene pelB. The genes pelB out of B.sub. and Xan. Code for pectinases or pectin lyases, yesZ being a beta-glucosidase, which could have enhanced the further dismanteling of the shell.
The BioBricks are supposed to be assembled performing a Gibson Assembly. For this we isolated the DNA of Bacillus subtilis and Xanthomonas campestris B100 and amplified the genes with specific primers. We wanted to clone the genes into psb1C3 and send them in. Using the E.coli vector pz9 availible to us in our lab we planned to characterize the genes. All in all we were trying to perform 6 clonings (3 clonings into psb1C3 and 3 into pz9). The psb1C3 constructs can be seen on the left. The experimentdesign was created with snapgene.
We also thought about using other, already in iGEM existing, enzymes:
Team Part Comments
WLC Milwaukee
2014 		BBa_K1175007
yesZ frim Bacillus Subtilis 		characterized in vitro assy
Statuts: It`s complicated
Edinburgh 2008 BBa_K118023
Codon optimized Caulobacter crescentus 		Caulobacter crescentus codon usage
British Columbia 2016 BBa_K2139003
Codon optimized Caulobacter crescentus 		sample not in stock
Endo-beta-1,4-glucanase E1
Stanford-Brown-Spelman 2014 BBa_K1499501
Endo-1,4-&-beta-glucanase (EG1) / Neisseria sicca 		Not characterized, but Neisseria sicca is an S2 organism
The experiments with liquid nitrogen and trypsin as well as the primer design strategy were especially supported by the supervisors. No antibiotics were handled by team members.

What went wrong?

Why didn’t the colonings work? Possible reasons for our failure are that we tried to perform a PCR using the wrong primers or that we committed an error somewhere else in the procedure. Another possibility is that we had the wrong stem of Bacillus subtilis from the beginning, because only the stem 168 has the required genes.
We also tried to order the parts for our clonings from IDT, but they were not able to send them to us in time for us to still work with them.