Difference between revisions of "Team:SHSID/Engineering"

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<h3>★  ALERT! </h3>
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<p>This page is used by the judges to evaluate your team for the <a href="https://2021.igem.org/Judging/Medals">medal criterion</a> or <a href="https://2021.igem.org/Judging/Awards"> award listed below</a>. </p>
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<h1>Engineering Success</h1>
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<h3> Silver Medal Criterion #1</h3>
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<p>Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle. This achievement should be distinct from your Contribution for Bronze.
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Please see the <a href="https://2021.igem.org/Judging/Medals">2021 Medals Page</a> for more information.
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<p>If you plan to show engineering success by creating a new Part that has been shown to work as expected, you must document your contribution on the Part's Main Page on the <a href="http://parts.igem.org/Main_Page">Registry</a> for your team to be eligible for this criteria.</p>
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    <div class="sub-content">
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        <div class="sub-title">Engineering</div>
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        <div class="article-title">Background </div>
 +
        <div class="article-content">Kl tropane alkaloids (TAs) refers to a kind of alkaloids containing the tropane
 +
            alkyl skeleton formed by the combination of pyrrole ring and piperidine ring in structure. It is a natural
 +
            product of plant and has a long history and important medicinal value. tropane alkaloids have great market
 +
            demand and often appear in global shortages. A method that can produce Tas in scale is expected. Using
 +
            synthetic biology to create a microbial cell factory to produce TAs is a highly potential strategy.</div>
 +
        <div class="article-title">Design</div>
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        <div class="article-content">The Tropine part of Tropane alkaloids (TAs) is obtained from arginine to putrescine
 +
            (1,4-butanediamine, putrescine), and then through a series of chemical reactions. In our project, natural
 +
            genes involved in arginine metabolism and polyamine biosynthesis was designed to overexpress in yeast. The
 +
            engineer strains that produced excess putrescine. The following genes were used to improve putrescine
 +
            production:</div>
 +
        <div class="article-content">(1) SPE1 gene: Overexpression of the SPE1 gene can increase the expression of Spe1p
 +
            which converts ornithine to putrescine.</div>
 +
        <div class="article-content">(2) AsADC and speB gene: Arginine is decarboxylated by arginine decarboxylase (ADC)
 +
            to agmatine (Agmatine), agmatine hydrolase ( AUH) can remove the guanidine group from agmatine and directly
 +
            produce putrescine. In this study, ADC from oats (AsADC) and AUH from E. coli (speB) were selected, both of
 +
            which have been shown to be active in Saccharomyces cerevisiae.</div>
 +
        <div class="article-title">Build </div>
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        <div class="article-content">The three genes SPE1, speB, and AsADC were inserted into yeast expression vector
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            pYES2. different promoters and terminators were applied for the transcription of the three genes,
 +
            respectively (Figure 1). </div>
 +
        <div class="img-wrap no-margin">
 +
            <img src="https://static.igem.org/mediawiki/2021/5/52/T--SHSID--engineering01.jpg" alt="" />
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            <span>Figure 2. DNA sequence map of plasmid pYES2-AsADC-SPE1-SpeB.</span>
 +
        </div>
 +
        <div class="article-title">Test </div>
 +
        <div class="article-content">Plasmids pYES2-AsADC-SPE1-SpeB were transferred to BY4741 chemically competent
 +
            yeast cells and screened on YPD/Hyg plates. Transformants were picked into YPD/Hyg medium for activation.
 +
            Metabolite production tests were carried out in YNB-SC fermentation medium (containing 10 times arginine raw
 +
            material). 48h metabolites (supernatant) were collected and the putrescine analyze metabolites by LC-MS.
 +
        </div>
 +
        <div class="img-wrap no-margin">
 +
            <img src="https://static.igem.org/mediawiki/2021/2/24/T--SHSID--engineering02.jpg" alt="" />
 +
            <span>Figure 3. The peak of putrescine detected by LC-MS.</span>
 +
        </div>
 +
        <div class="article-content">The results showed that at 32 min, the peak pattern of putrescine appeared (as
 +
            shown in the figure 3), indicating that the engineered strain we constructed successfully produced
 +
            putrescine. </div>
 +
        <div class="article-title">Learn</div>
 +
        <div class="article-content">The three genes that can improve putrescine production were successfully introduced
 +
            into the yeast strain. The yeast strain generates putrescine during fermentation experiment. What is
 +
            unsatisfactory is the peak area in LC-MS is small. The yield of putrescine is far below the factory mass
 +
            production level. It is likely that the protein expression is insufficient and the metabolic pathway is
 +
            small. In the future, the expression of the three proteins needs to be further optimized.</div>
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Revision as of 15:08, 18 October 2021

SHSID

Engineering
Background
Kl tropane alkaloids (TAs) refers to a kind of alkaloids containing the tropane alkyl skeleton formed by the combination of pyrrole ring and piperidine ring in structure. It is a natural product of plant and has a long history and important medicinal value. tropane alkaloids have great market demand and often appear in global shortages. A method that can produce Tas in scale is expected. Using synthetic biology to create a microbial cell factory to produce TAs is a highly potential strategy.
Design
The Tropine part of Tropane alkaloids (TAs) is obtained from arginine to putrescine (1,4-butanediamine, putrescine), and then through a series of chemical reactions. In our project, natural genes involved in arginine metabolism and polyamine biosynthesis was designed to overexpress in yeast. The engineer strains that produced excess putrescine. The following genes were used to improve putrescine production:
(1) SPE1 gene: Overexpression of the SPE1 gene can increase the expression of Spe1p which converts ornithine to putrescine.
(2) AsADC and speB gene: Arginine is decarboxylated by arginine decarboxylase (ADC) to agmatine (Agmatine), agmatine hydrolase ( AUH) can remove the guanidine group from agmatine and directly produce putrescine. In this study, ADC from oats (AsADC) and AUH from E. coli (speB) were selected, both of which have been shown to be active in Saccharomyces cerevisiae.
Build
The three genes SPE1, speB, and AsADC were inserted into yeast expression vector pYES2. different promoters and terminators were applied for the transcription of the three genes, respectively (Figure 1).
Figure 2. DNA sequence map of plasmid pYES2-AsADC-SPE1-SpeB.
Test
Plasmids pYES2-AsADC-SPE1-SpeB were transferred to BY4741 chemically competent yeast cells and screened on YPD/Hyg plates. Transformants were picked into YPD/Hyg medium for activation. Metabolite production tests were carried out in YNB-SC fermentation medium (containing 10 times arginine raw material). 48h metabolites (supernatant) were collected and the putrescine analyze metabolites by LC-MS.
Figure 3. The peak of putrescine detected by LC-MS.
The results showed that at 32 min, the peak pattern of putrescine appeared (as shown in the figure 3), indicating that the engineered strain we constructed successfully produced putrescine.
Learn
The three genes that can improve putrescine production were successfully introduced into the yeast strain. The yeast strain generates putrescine during fermentation experiment. What is unsatisfactory is the peak area in LC-MS is small. The yield of putrescine is far below the factory mass production level. It is likely that the protein expression is insufficient and the metabolic pathway is small. In the future, the expression of the three proteins needs to be further optimized.
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