Difference between revisions of "Team:CPU CHINA/Contribution"

 
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         <h1 id="title">CONTRIBUTIONS</h1>
 
         <h1 id="title">CONTRIBUTIONS</h1>
 
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         </div>
 
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         <div id="detail" class="clearfix">
 
         <div id="detail" class="clearfix">
 
             <div class="section" id="section1">
 
             <div class="section" id="section1">
 
                 <h2 id='overview-1'><span>OVERVIEW</span></h2>
 
                 <h2 id='overview-1'><span>OVERVIEW</span></h2>
−
                 <p><span> </span><span>Hoping to make a useful contribution for future iGEM teams, we completed the
+
                 <p>&#x200B; Hoping to make a <strong>useful contribution</strong> for future iGEM teams, we completed
−
                        experimental characterization of an existing part </span><strong><span>Ethanol regulated
+
                    the
−
                            promoter
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                    experimental characterization of an existing part <strong>Ethanol regulated promoter
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                            AOX1</span></strong>(<a href='http://parts.igem.org/Part:BBa_I764001' target='_blank'
+
                        AOX1</strong>(<a href="http://parts.igem.org/Part:BBa_I764001">BBa_I764001</a>) and
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                        class='url'>BBa_I764001</a>)<span> and provided new data for it.
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                    provided new data for it.</p>
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                    </span>
+
−
                </p>
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                 <figure>
 
                 <figure>
 
                     <table>
 
                     <table>
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                     </table>
 
                     </table>
 
                 </figure>
 
                 </figure>
−
                 <p><span>The details are in the part registrations page. </span></p>
+
                 <p>The <strong>details</strong> are in the part registrations page.</p>
 
             </div>
 
             </div>
 
             <div class="section" id="section2">
 
             <div class="section" id="section2">
 
                 <h2 id='contribution-2'><span>CONTRIBUTION</span></h2>
 
                 <h2 id='contribution-2'><span>CONTRIBUTION</span></h2>
−
                 <p><strong><span>P</span><sub><span>AOX1</span></sub></strong></p>
+
                 <h4><strong>P<sub>AOX1</sub></strong></h4>
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                 <p><span> </span><span>The AOX1 promoter（P</span><sub><span>AOX1</span></sub><span>） region from
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                 <p>&#x200B; The <strong>AOX1 promoter</strong>&#xFF08;P<sub>AOX1</sub>&#xFF09; region from <em>Pichia
−
                    </span><em><span>Pichia pastoris</span></em><span>. It was first registered in 2007 and used as a
+
                        pastoris</em>.
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                        strong
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                    It was first registered in 2007 and used as a strong promoter in <em>Pichia pastoris</em>. A complex
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                        promoter in </span><em><span>Pichia pastoris</span></em><span>. A complex pathway for the
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                    pathway for
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                        metabolism
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                    the metabolism of methanol exists within some species of the <em>Komagataella</em> genus.
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                        of methanol exists within some species of the </span><em><span>Komagataella</span></em><span>
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                    <strong>Alcohol oxidase
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                        genus.
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                        (AO)</strong> appears to be the first and major enzyme produced in this metabolic pathway.
−
                        Alcohol oxidase (AO) appears to be the first and major enzyme produced in this metabolic
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                    Transcribed from its
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                        pathway.
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                    gene (AOX1), AO converts <strong>methanol to formaldehyde</strong> within the yeast&apos;s
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                        Transcribed from its gene (AOX1), AO converts methanol to formaldehyde within the yeast&#39;s
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                    peroxisome.
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                        peroxisome.</span></p>
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                </p>
−
                 <p><span> </span><span>In order to test the function of P</span><sub><span>AOX1</span></sub><span>, we
+
                 <p>&#x200B; In order to test the function of P<sub>AOX1</sub>, we construct
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                        construct
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                    &quot;<strong>P<sub>AOX1</sub>-&#x3B1;-factor-sfGFP-AOX1
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                        &quot;</span><strong><span>P</span><sub><span>AOX1</span></sub><span>-α-factor-sfGFP-AOX1
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                        terminator&quot;(<a href="http://parts.igem.org/Part:BBa_K3853050">BBa_K3853050</a>)</strong>. If
−
                            terminator&quot;(<a href="http://parts.igem.org/Part:BBa_K3853050"
+
                    P<sub>AOX1</sub> is functional, we
−
                                target='_blank'>BBa_K3853050</a>)</span></strong><span>. If
+
                    can test the <strong>fluorescence intensity</strong> of sfGFP in <strong>supernatant
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                        P</span><sub><span>AOX1</span></sub><span> is functional, we can test the fluorescence intensity
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                        samples</strong> obtained
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                        of
+
                    from the culture of recombinant <em>P.pastoris</em> strain GS115.</p>
−
                        sfGFP in supernatant samples obtained from the culture of recombinant
+
                 <p>&#x200B; Our results matched the general expected trend (Fig 1). After fermentation experiment in
−
                    </span><em><span>P.pastoris</span></em><span> GS115 strain.</span></p>
+
                    BMMY medium
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                 <p>&nbsp;</p>
+
                    containing 0.5% methanol. The <strong>fluorescence intensity</strong> of the supernatant samples of
−
                <p><span> </span><span>Our results matched the general expected trend (Fig 1). After fermentation
+
                    recombinant
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                        experiment
+
                    <em>P.pastoris</em> strain GS115 containing the sfGFP gene gradually <strong>increased over time</strong>,
−
                        in BMMY medium containing 0.5% methanol. The fluorescence intensity of the supernatant samples
+
                    while that of
−
                        of
+
                    wild-type <em>P.pastoris</em> strain GS115 remained <strong>basically unchanged</strong>, which is in line
−
                        recombinant </span><em><span>P.pastoris</span></em><span> GS115 containing the sfGFP gene
+
                    with
−
                        gradually
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                    literature description<sup>[1]</sup>. SDS-PAGE results (Fig 2) also verified this phenomenon, almost
−
                        increased over time, while that of wild-type </span><em><span>P.pastoris</span></em><span> GS115
+
                    no protein
−
                        remained basically unchanged, which is in line with literature
+
                    band before 36 h could be seen. The corresponding protein band <strong>began to appear</strong> at
−
                        description</span><sup><span>[1]</span></sup><span>. SDS-PAGE results (Fig 2) also verified this
+
                    36 h, and the
−
                        phenomenon, almost no protein band before 36 h could be seen. The corresponding protein band
+
                    clarity and width of the protein band gradually increased over time, which means the AOX1 promoter
−
                        began
+
                    can
−
                        to appear at 36 h, and the clarity and width of the protein band gradually increased over time,
+
                    continuously induce the expression of the protein. At the same time, by measuring <strong>the growth
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                        which means the AOX1 promoter can continuously induce the expression of the protein. At the same
+
                        curve</strong> of the strains (Fig 3), we observed that the OD<sub>600</sub> of the recombinant
−
                        time, by measuring the growth curve of the strains (Fig 3), we observed that the
+
                     <em>P.pastoris</em> strain GS115 containing the sfGFP gene was <strong>slightly lower</strong> than the
−
                        OD</span><sub><span>600</span></sub><span> of the recombinant
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                    wild-type
−
                     </span><em><span>P.pastoris</span></em><span> GS115 containing the sfGFP gene was slightly lower
+
                    <em>P.pastoris</em> strain GS115 , the phenomenon of which may be attributed to the expression of sfGFP.
−
                        than
+
                    The results
−
                        the wild-type </span><em><span>P.pastoris</span></em><span> GS115 , the phenomenon of which may
+
                    showed that the expression of foreign genes would <strong>partly inhibit cell growth</strong>, but
−
                        be
+
                    not in an
−
                        attributed to the expression of sfGFP. The results showed that the expression of foreign genes
+
                    intensive manner.
−
                        would
+
                </p>
−
                        partly inhibit cell growth, but not in an intensive manner.</span></p>
+
                <img src="https://static.igem.org/mediawiki/2021/7/72/T--CPU_CHINA--Parts--Contributions--fig1.png"
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                 <p><img src="https://static.igem.org/mediawiki/2021/7/72/T--CPU_CHINA--Parts--Contributions--fig1.png"
+
                        referrerpolicy="no-referrer">
 +
                 <p class="imgdescribe"><strong><span>Fig. 1 Fluorescence intensity of supernatant samples obtained at
 +
                        different time points from
 +
                        the
 +
                        culture of wild-type <em>P.pastoris</em> strain GS115 and corresponding recombinant <em>P.pastoris</em> strain
 +
                        GS115
 +
                        containing
 +
                        sfGFP gene.</span></strong></p>
 +
                <img src="https://static.igem.org/mediawiki/2021/f/f2/T--CPU_CHINA--Parts--Contributions--fig2.png"
 
                         referrerpolicy="no-referrer"></p>
 
                         referrerpolicy="no-referrer"></p>
−
                 <p class="imgdescribe"><span>Fig. 1 Fluorescence intensity of supernatant samples obtained at
+
                 <p class="imgdescribe"><strong><span>Fig. 2 SDS-PAGE gel analysis of supernatant samples of the recombinant
−
                            different time points from
+
                        <em>P.pastoris</em> strain GS115
−
                            the
+
                        containing the sfGFP gene during the fermentation</span></strong></p>
−
                            culture of wild-type <em>P.pastoris</em> GS115 and corresponding recombinant <em>P.pastoris</em> GS115
+
−
                            containing
+
−
                            sfGFP gene.</span></p>
+
−
                <p><img src="https://static.igem.org/mediawiki/2021/f/f2/T--CPU_CHINA--Parts--Contributions--fig2.png"
+
−
                        referrerpolicy="no-referrer"></p>
+
−
                <p class="imgdescribe"><span>Fig. 2 SDS-PAGE gel analysis of supernatant samples of the recombinant
+
−
                    <em>P.pastoris</em> GS115
+
−
                            containing the sfGFP gene during the fermentation</span></p>
+
 
                 <p>&nbsp;</p>
 
                 <p>&nbsp;</p>
−
                 <p><img src="https://static.igem.org/mediawiki/2021/4/44/T--CPU_CHINA--Parts--Contributions--fig3.png"
+
                 <img src="https://static.igem.org/mediawiki/2021/4/44/T--CPU_CHINA--Parts--Contributions--fig3.png"
−
                         referrerpolicy="no-referrer"></p>
+
                         referrerpolicy="no-referrer">
−
                 <p class="imgdescribe"><span>Fig. 3 OD</span><sub><span>600</span></sub><span> absorbance obtained at
+
                 <p class="imgdescribe"><strong><span>Fig. 3 OD</span><sub><span>600</span></sub><span> absorbance obtained at
−
                            different time
+
                        different time
−
                            points
+
                        points
−
                            from the culture of wild-type <em>P.pastoris</em> GS115 and recombinant <em>P.pastoris</em> GS115 that
+
                        from the culture of wild-type <em>P.pastoris</em> strain GS115 and recombinant <em>P.pastoris</em> strain
−
                            contains
+
                        GS115 that
−
                            sfGFP gene</span><span>.</span></p>
+
                        contains
−
                 <p><strong><span>Reference</span></strong></p>
+
                        sfGFP gene</span><span>.</span></strong></p>
 +
                 <h4><strong><span>Reference</span></strong></h4>
 
                 <p class="reference"><span>[1] Xuan, Y. </span><em><span>et al.</span></em><span> An upstream activation
 
                 <p class="reference"><span>[1] Xuan, Y. </span><em><span>et al.</span></em><span> An upstream activation
 
                         sequence controls
 
                         sequence controls
 
                         the
 
                         the
 
                         expression of AOX1 gene in Pichia pastoris. </span><em><span>FEMS yeast
 
                         expression of AOX1 gene in Pichia pastoris. </span><em><span>FEMS yeast
−
                             research</span></em><span>,
+
                             research</span></em> <span><strong>9</strong>,
 
                         1271-1282, doi:10.1111/j.1567-1364.2009.00571.x (2009).</span></p>
 
                         1271-1282, doi:10.1111/j.1567-1364.2009.00571.x (2009).</span></p>
 
             </div>
 
             </div>
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Latest revision as of 04:04, 22 October 2021

OVERVIEW

​ Hoping to make a useful contribution for future iGEM teams, we completed the experimental characterization of an existing part Ethanol regulated promoter AOX1(BBa_I764001) and provided new data for it.

Biobricks codes in the lab Quantitative Characterization
BBa_I764001 promoter fluorescence intensity

The details are in the part registrations page.

CONTRIBUTION

PAOX1

​ The AOX1 promoter（PAOX1） region from Pichia pastoris. It was first registered in 2007 and used as a strong promoter in Pichia pastoris. A complex pathway for the metabolism of methanol exists within some species of the Komagataella genus. Alcohol oxidase (AO) appears to be the first and major enzyme produced in this metabolic pathway. Transcribed from its gene (AOX1), AO converts methanol to formaldehyde within the yeast's peroxisome.

​ In order to test the function of PAOX1, we construct "PAOX1-α-factor-sfGFP-AOX1 terminator"(BBa_K3853050). If PAOX1 is functional, we can test the fluorescence intensity of sfGFP in supernatant samples obtained from the culture of recombinant P.pastoris strain GS115.

​ Our results matched the general expected trend (Fig 1). After fermentation experiment in BMMY medium containing 0.5% methanol. The fluorescence intensity of the supernatant samples of recombinant P.pastoris strain GS115 containing the sfGFP gene gradually increased over time, while that of wild-type P.pastoris strain GS115 remained basically unchanged, which is in line with literature description[1]. SDS-PAGE results (Fig 2) also verified this phenomenon, almost no protein band before 36 h could be seen. The corresponding protein band began to appear at 36 h, and the clarity and width of the protein band gradually increased over time, which means the AOX1 promoter can continuously induce the expression of the protein. At the same time, by measuring the growth curve of the strains (Fig 3), we observed that the OD600 of the recombinant P.pastoris strain GS115 containing the sfGFP gene was slightly lower than the wild-type P.pastoris strain GS115 , the phenomenon of which may be attributed to the expression of sfGFP. The results showed that the expression of foreign genes would partly inhibit cell growth, but not in an intensive manner.

Fig. 1 Fluorescence intensity of supernatant samples obtained at different time points from the culture of wild-type P.pastoris strain GS115 and corresponding recombinant P.pastoris strain GS115 containing sfGFP gene.

Fig. 2 SDS-PAGE gel analysis of supernatant samples of the recombinant P.pastoris strain GS115 containing the sfGFP gene during the fermentation

 

Fig. 3 OD600 absorbance obtained at different time points from the culture of wild-type P.pastoris strain GS115 and recombinant P.pastoris strain GS115 that contains sfGFP gene.

Reference

[1] Xuan, Y. et al. An upstream activation sequence controls the expression of AOX1 gene in Pichia pastoris. FEMS yeast research 9, 1271-1282, doi:10.1111/j.1567-1364.2009.00571.x (2009).