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

 
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<h1>Parts</h1>
  
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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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<h2>New Parts Submitted to the Registry in 2016 </h2>
  
 
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<h4>Composite Part 1: BBa_K2185003</h4>
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<h3>Composite Part 1: BBa_K2185003</h3>
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Promoterless
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<p1>Promoterless</p1>
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Kozak sequence (BBa_K165002)
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<p1>Kozak sequence (BBa_K165002)</p1>
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Mating Factor Secretion Tag (BBa_K792002)
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<p1>Mating Factor Secretion Tag (BBa_K792002)</p1>
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Alpha amylase coding sequence from the fungus Penicillium  
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<p1>Alpha amylase coding sequence from the fungus Penicillium </p1>
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ADH1 Terminator (Part BBa_K392003)
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<p1>ADH1 Terminator (Part BBa_K392003)</p1>
  
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<h4>Composite Part 2: BBa_K2185005</h4>
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<h3>Composite Part 2: BBa_K2185005</h3>
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Promoterless
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<p1>Promoterless</p1>
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Kozak sequence (Part BBa_K165002)
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<p1>Kozak sequence (Part BBa_K165002)</p1>
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Mating Factor Secretion Tag (BBa_K792002)
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<p1>Mating Factor Secretion Tag (BBa_K792002)</p1>
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Beta amylase coding sequence from Bacillus cereus
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<p1>Beta amylase coding sequence from Bacillus cereus</p1>
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ADH1 Terminator (Part BBa_K392003)
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<p1>ADH1 Terminator (Part BBa_K392003)</p1>
  
  
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<h4>Composite Part 3: BBa_K2185007</h4>
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<h3>Composite Part 3: BBa_K2185007</h3>
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pCyc medium promoter (BBa_I766555)
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<p1>pCyc medium promoter (BBa_I766555)</p1>
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Kozak sequence (Part BBa_K165002)
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<p1>Kozak sequence (Part BBa_K165002)</p1>
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Mating Factor Secretion Tag (BBa_K792002)
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<p1>Mating Factor Secretion Tag (BBa_K792002)</p1>
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Alpha amylase coding sequence from Saprolegnia ferax
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<p1>Alpha amylase coding sequence from Saprolegnia ferax</p1>
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ADH1 Terminator (Part BBa_K392003)
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<p1>ADH1 Terminator (Part BBa_K392003)</p1>
 
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<h4><u>Description of Basic Parts:</u> </h4>
 
  
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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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<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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<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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<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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<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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<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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<h3>Improvement in Characterization of Part from 2015 </h3>
 
  
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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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<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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<h4><u>Description of Basic Parts:</u> </h4>
  
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<p1><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.</p1>  
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[[File:Brhs_visualstarchdegradation.png|500px]]
 
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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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<p1><b>Kozak Sequence</b> - This part initiates translation from eukaryotic mRNA and is placed between a promoter and coding sequence to facilitate translation. </p1>
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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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<b>pCyc medium promoter</b> - This is a medium expression level constitutive promoter. It directs the cell to transcribe the DNA sequence constantly. </p1>
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<br><br>
  
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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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<p1>
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</p>
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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. </p1>
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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.  </p1>
 +
<br><br>
  
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<p1>
 +
<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.  </p1>
 +
<br><br>
  
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<p1>
 +
<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. </p1>
 +
<br><br>
  
  
 +
<p1>In addition to the 3 new parts submitted this year, we also improved a previous part, from last year. To see how we improved the characterization of this part, please see <a href="http://parts.igem.org/Part:BBa_K1871002">here</a>. </p1>
 
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Latest revision as of 19:49, 19 October 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.

In addition to the 3 new parts submitted this year, we also improved a previous part, from last year. To see how we improved the characterization of this part, please see here.