Difference between revisions of "Team:BroadRun-Baltimore/Parts"

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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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<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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<h1>Parts </h1>
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<h3>New Parts Submitted to the Registry in 2016 </h3>
  
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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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    <th>Part Name</th>
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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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    <th>Part Number</th>
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    <td>Penicillium alpha amylase w/ no promoter</td>
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    <td>BBa_K2185003</td>
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    <td>B.cereus beta amylase w/ no promoter</td>
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    <td>BBa_K2185005</td>
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    <td>S.ferax alpha amylase w/ cyc promoter</td>
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    <td>BBa_K2185007</td>
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<h4>Composite Part 1: BBa_K2185003</h4>
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Promoterless
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Kozak sequence (BBa_K165002)
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Mating Factor Secretion Tag (BBa_K792002)
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Alpha amylase coding sequence from the fungus Penicillium
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ADH1 Terminator (Part BBa_K392003)
  
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<h4>Composite Part 2: BBa_K2185005</h4>
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Promoterless
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Kozak sequence (Part BBa_K165002)
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Mating Factor Secretion Tag (BBa_K792002)
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Beta amylase coding sequence from Bacillus cereus
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ADH1 Terminator (Part BBa_K392003)
  
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<h5>What information do I need to start putting my parts on the Registry?</h5>
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<h4>Composite Part 3: BBa_K2185007</h4>
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<p>The information needed to initially create a part on the Registry is:</p>
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pCyc medium promoter (BBa_I766555)
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<ul>
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Kozak sequence (Part BBa_K165002)
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<li>Part Name</li>
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Mating Factor Secretion Tag (BBa_K792002)
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<li>Part type</li>
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Alpha amylase coding sequence from Saprolegnia ferax
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<li>Creator</li>
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ADH1 Terminator (Part BBa_K392003)
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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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<h4><u>Description of Basic Parts:</u> </h4>
  
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<h5>Inspiration</h5>
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<b>ADH1 Terminator</b> - This part is the terminator region from yeast alcohol dehydrogenase (ADH1) gene. This stops the RNA polymerase from transcribing the DNA sequence.
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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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<br><br>
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<b>Kozak Sequence</b> - This part initiates translation from eukaryotic mRNA and is placed between a promoter and coding sequence to facilitate translation.
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<br><br>
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<b>pCyc medium promoter</b> - This is a medium expression level constitutive promoter. It directs the cell to transcribe the DNA sequence constantly.
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<br><br>
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<b>Mating Factor Alpha Secretion Sequence</b> - This part is the secretion signal from yeast α-mating factor, and directs the secretion of the produced protein. This allows the exportation of the protein.  
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<br><br>
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<b>Penicillium Alpha Amylase</b> - Obtained from the fungus Penicillium, α-Amylase is an enzyme that hydrolyses alpha bonds of large, alpha-linked polysaccharides, such as starch and glycogen, thus degrading the polysaccharides into monosaccharides and disaccharides. 
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<br><br>
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<b>S.ferax Alpha Amylase</b> - Obtained from the fungus Saprolegnia ferax, α-Amylase is an enzyme that hydrolyses alpha bonds of large, alpha-linked polysaccharides, such as starch and glycogen, thus degrading the polysaccharides into monosaccharides and disaccharides.
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<br><br>
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<b>B.cereus Beta Amylase</b> - Obtained from the fungus Saprolegnia ferax, α-Amylase is an enzyme that hydrolyses beta bonds of large, beta-linked polysaccharides, such as cellulose and some starches, thus degrading the polysaccharides into monosaccharides and disaccharides.
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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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<h3>Improvement in Characterization of Part from 2015 </h3>
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<ul>
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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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<p>In 2015, we designed and synthesized 3 composite parts (link), but were only able to test one of them for proof of concept. This was because we did not succeed in cloning the other two composite parts into the yeast vector, thus we were not able to get the composite part into yeast and test it. This year, we decided to try again to get those composite parts into yeast. To do so, we changed the restriction enzymes and gel extracted the yeast plasmid (instead of a DNA purification). The gel extraction was to prevent the plasmid from ligating back on itself, without the insert inside the plasmid.
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<h5>Part Table </h5>
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<p>We succeeded in cloning one of our composite parts from last year into yeast, Composite Part (BBa_K1871002). We then tested the genetically modified yeast containing this composite part. We were able to confirm that the yeast were expressing the alpha amylase enzyme by showing starch degradation over a period of 6 hours. Our results are shown below. </p>
  
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<p><i>Figure 1.</i> The image above was taken 5 hours after adding the genetically modified yeast to a starch solution. The yeast strain containing a plasmid with this composite part is the third tube from the left. The right most tube is the control, containing wildtype yeast and starch. Iodine, which reacts with starch to form a blue color, was added to each solution to determine the amount of starch. The third tube from the left has a very light blue color, indicating a small amount of starch left. The right most tube has a dark blue color, indicating a large amount of starch left. This shows that the yeast cells are producing and secreting amylase enzymes, thus the composite part is fully functional.</p></html>
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<p><i>Figure 2.</i> The image above shows a quantification of starch degradation over a period of 6 hours. A spectrophotometer was used to quantify the amount of blue color in each tube, seen in the figure above. An absorbance level of 1 equals a starch concentration of 0.25%. The yeast strain with this composite part is labeled as "Modified Yeast Strain 3" in the graph. For information on our results please see our <a href="https://2016.igem.org/Team:BroadRun-Baltimore/Parts">Results</a> page. </p>
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<p>With these new results, we were able to improve the characterization of this part. The newly characterized part can be seen on the <a href="http://parts.igem.org/Part:BBa_K1871002">Registry of Parts</a>.
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Revision as of 21:17, 5 September 2016

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Parts


New Parts Submitted to the Registry in 2016

Part Name Part Number
Penicillium alpha amylase w/ no promoter BBa_K2185003
B.cereus beta amylase w/ no promoter BBa_K2185005
S.ferax alpha amylase w/ cyc promoter BBa_K2185007

Composite Part 1: BBa_K2185003

Promoterless Kozak sequence (BBa_K165002) Mating Factor Secretion Tag (BBa_K792002) Alpha amylase coding sequence from the fungus Penicillium ADH1 Terminator (Part BBa_K392003)

Composite Part 2: BBa_K2185005

Promoterless Kozak sequence (Part BBa_K165002) Mating Factor Secretion Tag (BBa_K792002) Beta amylase coding sequence from Bacillus cereus ADH1 Terminator (Part BBa_K392003)

Composite Part 3: BBa_K2185007

pCyc medium promoter (BBa_I766555) Kozak sequence (Part BBa_K165002) Mating Factor Secretion Tag (BBa_K792002) Alpha amylase coding sequence from Saprolegnia ferax ADH1 Terminator (Part BBa_K392003)

Description of Basic Parts:

ADH1 Terminator - This part is the terminator region from yeast alcohol dehydrogenase (ADH1) gene. This stops the RNA polymerase from transcribing the DNA sequence.

Kozak Sequence - This part initiates translation from eukaryotic mRNA and is placed between a promoter and coding sequence to facilitate translation.

pCyc medium promoter - This is a medium expression level constitutive promoter. It directs the cell to transcribe the DNA sequence constantly.

Mating Factor Alpha Secretion Sequence - This part is the secretion signal from yeast α-mating factor, and directs the secretion of the produced protein. This allows the exportation of the protein.

Penicillium Alpha Amylase - Obtained from the fungus Penicillium, α-Amylase is an enzyme that hydrolyses alpha bonds of large, alpha-linked polysaccharides, such as starch and glycogen, thus degrading the polysaccharides into monosaccharides and disaccharides.

S.ferax Alpha Amylase - Obtained from the fungus Saprolegnia ferax, α-Amylase is an enzyme that hydrolyses alpha bonds of large, alpha-linked polysaccharides, such as starch and glycogen, thus degrading the polysaccharides into monosaccharides and disaccharides.

B.cereus Beta Amylase - Obtained from the fungus Saprolegnia ferax, α-Amylase is an enzyme that hydrolyses beta bonds of large, beta-linked polysaccharides, such as cellulose and some starches, thus degrading the polysaccharides into monosaccharides and disaccharides.

Improvement in Characterization of Part from 2015

In 2015, we designed and synthesized 3 composite parts (link), but were only able to test one of them for proof of concept. This was because we did not succeed in cloning the other two composite parts into the yeast vector, thus we were not able to get the composite part into yeast and test it. This year, we decided to try again to get those composite parts into yeast. To do so, we changed the restriction enzymes and gel extracted the yeast plasmid (instead of a DNA purification). The gel extraction was to prevent the plasmid from ligating back on itself, without the insert inside the plasmid.

We succeeded in cloning one of our composite parts from last year into yeast, Composite Part (BBa_K1871002). We then tested the genetically modified yeast containing this composite part. We were able to confirm that the yeast were expressing the alpha amylase enzyme by showing starch degradation over a period of 6 hours. Our results are shown below.


500px

Figure 1. The image above was taken 5 hours after adding the genetically modified yeast to a starch solution. The yeast strain containing a plasmid with this composite part is the third tube from the left. The right most tube is the control, containing wildtype yeast and starch. Iodine, which reacts with starch to form a blue color, was added to each solution to determine the amount of starch. The third tube from the left has a very light blue color, indicating a small amount of starch left. The right most tube has a dark blue color, indicating a large amount of starch left. This shows that the yeast cells are producing and secreting amylase enzymes, thus the composite part is fully functional.

500px

Figure 2. The image above shows a quantification of starch degradation over a period of 6 hours. A spectrophotometer was used to quantify the amount of blue color in each tube, seen in the figure above. An absorbance level of 1 equals a starch concentration of 0.25%. The yeast strain with this composite part is labeled as "Modified Yeast Strain 3" in the graph. For information on our results please see our Results page.


With these new results, we were able to improve the characterization of this part. The newly characterized part can be seen on the Registry of Parts.


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