Team:Alberta/Achievements/Overview

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         <h2>Proof of Concept</h2>
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         <h2>Overview</h2>
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        <img src="https://static.igem.org/mediawiki/2011/0/03/Overview-pic.png" width=300px style="float:right;margin:20px">
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        <h3>Growth</h3>
 
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        <p>We wanted to see how well <i>N. crassa</i> grew on cellulosic media. We tested using grass clippings and wheat straw. Race tubes were used to determine the rate of hyphal growth of <i>N. Crassa</i> with different growth mediums. The following are graphs of the results:</p>
 
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        <p>Team Alberta’s divided approach allowed for simultaneous production of methods to characterize and lay the foundations for a unified project. Our plan was to use cellulose by-products to produce a viable fuel. The questions we sought to answer were: </p>
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            <li>What organism can we utilize that already consumes cellulose?</li>
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            <li>How can this organism be modified to increase its fatty acid content? </li>
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            <li>How can we make genetic modifications easy and accessible for this organism?</li>
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            <li>How can we use our organism’s outputs to make a viable fuel?</li>
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            <li>How could our biodiesel affect populations?</li>
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         <p>The <a href="https://2011.igem.org/Team:Alberta/Growth">growth section</a> of our project successfully conveyed <i>N. crassa's</i> ability to utilize cellulose by using grass clippings and wheat straw as characterized examples. We found the growth rates of <i>Neurospora</i> on these substrates comparable to that of <i>Neurospora</i> on Vogel's media (VSuTB).</p>
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        <p>Our project aimed at disrupting beta-oxidation (fatty acid breakdown) and upregulating fatty acid synthesis. We aimed to accomplish this by inserting a thioesterase into the fatty acyl CoA synthetase gene site of <i>Neurospora</i>, thereby simultaneously knocking out an essential gene to beta oxidation and inserting a gene to aide in fatty acid synthesis. We soon learned that constructing genes for <i>Neurospora</i> was not a well-defined or simple process, which is why Team Alberta spent the majority of the summer developing a rapid method to create genes for <i>Neurospora</i> based on our previous experiences in gene construction. These innovations lead to a new assembly method and the creation of <a href="https://static.igem.org/mediawiki/2011/a/ae/RFC-82.pdf">BBF RFC 82</a>. </p>
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            <img src="https://static.igem.org/mediawiki/2011/4/4c/Proofofconceptgraph2.png" width=600px>
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        <p>An important aspect to our project was achieving a usable fuel. We developed an efficient and direct method of <a href="https://2011.igem.org/Team:Alberta/EsterificationExtraction">chemical esterification</a> to effectively allow the fatty acids of <i>Neurospora</i> to be changed into fatty acid methyl esters, an accepted form of biodiesel. We were able to characterize the output of <i>Neurospora</i> fatty acid production as mainly C16 and C18 fatty acids.</p>
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         <p>Using our data from the wild type <i>Neurospora crassa</i>, we conducted a <a href="https://2011.igem.org/Team:Alberta/HumanPractices/CostAnalysis">cost analysis</a> to determine how economical our biodiesel would be and achieved great results for the average savings in cities around the world.</p>
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         <p>The graphs show similar growth rates for the grass and wheat straw as compared to the minimal media control VsuTB. This demonstrates that <i>N. crassa</i> does grow well on cellulosic biomass which makes it a good organism to use for large scale production of biodiesel. </p>
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         <p>Team Alberta has worked hard over the summer and is proud to be presenting our multifaceted project. We are excited to be introducing <i>Neurospora crassa</i> to the iGEM community and to share our many achievements in the development in this organism. </p>
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        <h3>Genetics</h3>
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         <p>The multiple assembly approach as described in the <a href="">RFC</a> were tested to determine the efficiency and fidelity of the assembly method. A key requirement of this approach is that upon ligation, ends must find partners precisely without promiscuity. To test this requirement and select ends that meet this criterion we subdivided the coding sequence of the alpha subunit of beta galactosidase into nine segments ending four base 5' overhangs that were selected to eliminate duplication Also included were two segments encoding a promoter (Bba J23001) and one segment encoding the transcriptional terminator Bba 1002 as shown below:</p>
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            <img src="https://static.igem.org/mediawiki/2011/9/95/Alberta-Sequence.png">
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        <p>Segments were produced from desalted phosphorylated oligonucleotides (IDT) that had been annealed over a two-hour temperature gradient from 70 to 25 degrees. Plasmids that contained the BetaGal cassette were then assembled using the biobytes sequential assembly method on magnetic beads as described by the Alberta iGEM 2010 team. Briefly, all segments were mixed and ligated degrees. simultaneously at 50-fold molar excess to a bead-bound origin of replication derived from pSB1C3 at the restrictive temperatureof 37. Following several washes, the KanR gene was ligated and capped for recircularization. The construct was then released from the beads and to avoid bias in colony selection, transformants were plated onto media lacking XGal. Ten colonies were then selected for sequencing. Notably, the DNA from all ten colonies contained the complete alpha-BetaGal cassette attesting to the precision of the biobyte assembly method. Only one -1 frameshift was detected in the first promoter segment representing an accuracy of synthesis of 1 mistake in 3000 base pairs. The fact the no mutations were detected at the boundaries of segment ligation illustrates that any of the overhangs defined above were suitable for the <i>N. crassa</i> gene insertion design described above.</p>
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        <h3>Esterification</h3>
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        <p><i>N. crassa</i> was esterified directly and the fatty acids were analyzed using GC-MS, an example of the GC plot is given below:</p>
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        <p>This shows that <i>N. crassa</i> can be esterified using our direct esterification method to produce our biodiesel, which are fatty acid methyl esters, especially the C16 fatty acids which are expected to accumulate as a result of our genetic manipulations. </p>
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Latest revision as of 03:04, 29 September 2011

ACHIEVEMENTS

Overview

Team Alberta’s divided approach allowed for simultaneous production of methods to characterize and lay the foundations for a unified project. Our plan was to use cellulose by-products to produce a viable fuel. The questions we sought to answer were:

  1. What organism can we utilize that already consumes cellulose?
  2. How can this organism be modified to increase its fatty acid content?
  3. How can we make genetic modifications easy and accessible for this organism?
  4. How can we use our organism’s outputs to make a viable fuel?
  5. How could our biodiesel affect populations?

The growth section of our project successfully conveyed N. crassa's ability to utilize cellulose by using grass clippings and wheat straw as characterized examples. We found the growth rates of Neurospora on these substrates comparable to that of Neurospora on Vogel's media (VSuTB).


Our project aimed at disrupting beta-oxidation (fatty acid breakdown) and upregulating fatty acid synthesis. We aimed to accomplish this by inserting a thioesterase into the fatty acyl CoA synthetase gene site of Neurospora, thereby simultaneously knocking out an essential gene to beta oxidation and inserting a gene to aide in fatty acid synthesis. We soon learned that constructing genes for Neurospora was not a well-defined or simple process, which is why Team Alberta spent the majority of the summer developing a rapid method to create genes for Neurospora based on our previous experiences in gene construction. These innovations lead to a new assembly method and the creation of BBF RFC 82.


An important aspect to our project was achieving a usable fuel. We developed an efficient and direct method of chemical esterification to effectively allow the fatty acids of Neurospora to be changed into fatty acid methyl esters, an accepted form of biodiesel. We were able to characterize the output of Neurospora fatty acid production as mainly C16 and C18 fatty acids.


Using our data from the wild type Neurospora crassa, we conducted a cost analysis to determine how economical our biodiesel would be and achieved great results for the average savings in cities around the world.


Team Alberta has worked hard over the summer and is proud to be presenting our multifaceted project. We are excited to be introducing Neurospora crassa to the iGEM community and to share our many achievements in the development in this organism.

About our Wiki

Seven students from three departments across the University of Alberta campus aspired to complete an ambitious project over the course of the summer. This wiki is all about our successes and our many trials and tribulations.

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University of Alberta

Edmonton, Alberta T6G 2E1


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