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<div id="detail" class="clearfix"> | <div id="detail" class="clearfix"> | ||
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<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> | ||
− | < | + | <h4><strong>P<sub>AOX1</sub></strong></h4> |
<p>​ The <strong>AOX1 promoter</strong>(P<sub>AOX1</sub>) region from <em>Pichia | <p>​ The <strong>AOX1 promoter</strong>(P<sub>AOX1</sub>) region from <em>Pichia | ||
pastoris</em>. | pastoris</em>. | ||
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<p>​ In order to test the function of P<sub>AOX1</sub>, we construct | <p>​ In order to test the function of P<sub>AOX1</sub>, we construct | ||
"<strong>P<sub>AOX1</sub>-α-factor-sfGFP-AOX1 | "<strong>P<sub>AOX1</sub>-α-factor-sfGFP-AOX1 | ||
− | terminator"(<a href="http://parts.igem.org/Part: | + | terminator"(<a href="http://parts.igem.org/Part:BBa_K3853050">BBa_K3853050</a>)</strong>. If |
P<sub>AOX1</sub> is functional, we | P<sub>AOX1</sub> is functional, we | ||
can test the <strong>fluorescence intensity</strong> of sfGFP in <strong>supernatant | can test the <strong>fluorescence intensity</strong> of sfGFP in <strong>supernatant | ||
samples</strong> obtained | samples</strong> obtained | ||
− | from the culture of recombinant <em>P.pastoris</em> | + | from the culture of recombinant <em>P.pastoris</em> strain GS115.</p> |
<p>​ Our results matched the general expected trend (Fig 1). After fermentation experiment in | <p>​ Our results matched the general expected trend (Fig 1). After fermentation experiment in | ||
BMMY medium | BMMY medium | ||
containing 0.5% methanol. The <strong>fluorescence intensity</strong> of the supernatant samples of | containing 0.5% methanol. The <strong>fluorescence intensity</strong> of the supernatant samples of | ||
recombinant | recombinant | ||
− | <em>P.pastoris</em> GS115 containing the sfGFP gene gradually <strong>increased over time</strong>, | + | <em>P.pastoris</em> strain GS115 containing the sfGFP gene gradually <strong>increased over time</strong>, |
while that of | while that of | ||
− | wild-type <em>P.pastoris</em> GS115 remained <strong>basically unchanged</strong>, which is in line | + | wild-type <em>P.pastoris</em> strain GS115 remained <strong>basically unchanged</strong>, which is in line |
with | with | ||
literature description<sup>[1]</sup>. SDS-PAGE results (Fig 2) also verified this phenomenon, almost | literature description<sup>[1]</sup>. SDS-PAGE results (Fig 2) also verified this phenomenon, almost | ||
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continuously induce the expression of the protein. At the same time, by measuring <strong>the growth | continuously induce the expression of the protein. At the same time, by measuring <strong>the growth | ||
curve</strong> of the strains (Fig 3), we observed that the OD<sub>600</sub> of the recombinant | curve</strong> of the strains (Fig 3), we observed that the OD<sub>600</sub> of the recombinant | ||
− | <em>P.pastoris</em> GS115 containing the sfGFP gene was <strong>slightly lower</strong> than the | + | <em>P.pastoris</em> strain GS115 containing the sfGFP gene was <strong>slightly lower</strong> than the |
wild-type | wild-type | ||
− | <em>P.pastoris</em> GS115 , the phenomenon of which may be attributed to the expression of sfGFP. | + | <em>P.pastoris</em> strain GS115 , the phenomenon of which may be attributed to the expression of sfGFP. |
The results | The results | ||
showed that the expression of foreign genes would <strong>partly inhibit cell growth</strong>, but | showed that the expression of foreign genes would <strong>partly inhibit cell growth</strong>, but | ||
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different time points from | different time points from | ||
the | the | ||
− | culture of wild-type <em>P.pastoris</em> GS115 and corresponding recombinant <em>P.pastoris</em> | + | culture of wild-type <em>P.pastoris</em> strain GS115 and corresponding recombinant <em>P.pastoris</em> strain |
GS115 | GS115 | ||
containing | containing | ||
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referrerpolicy="no-referrer"></p> | referrerpolicy="no-referrer"></p> | ||
<p class="imgdescribe"><strong><span>Fig. 2 SDS-PAGE gel analysis of supernatant samples of the recombinant | <p class="imgdescribe"><strong><span>Fig. 2 SDS-PAGE gel analysis of supernatant samples of the recombinant | ||
− | <em>P.pastoris</em> GS115 | + | <em>P.pastoris</em> strain GS115 |
containing the sfGFP gene during the fermentation</span></strong></p> | containing the sfGFP gene during the fermentation</span></strong></p> | ||
<p> </p> | <p> </p> | ||
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different time | different time | ||
points | points | ||
− | from the culture of wild-type <em>P.pastoris</em> GS115 and recombinant <em>P.pastoris</em> | + | from the culture of wild-type <em>P.pastoris</em> strain GS115 and recombinant <em>P.pastoris</em> strain |
GS115 that | GS115 that | ||
contains | contains | ||
sfGFP gene</span><span>.</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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</li> | </li> | ||
<li> | <li> | ||
− | <a href="cpuchina2021@163.com"> | + | <a href="mailto:cpuchina2021@163.com"> |
<img src="https://static.igem.org/mediawiki/2021/9/92/T--CPU_CHINA--emailLogo.png" style="margin-bottom: 2vw;" alt=""> | <img src="https://static.igem.org/mediawiki/2021/9/92/T--CPU_CHINA--emailLogo.png" style="margin-bottom: 2vw;" alt=""> | ||
<p>cpuchina2021@163.com</p> | <p>cpuchina2021@163.com</p> | ||
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+ | <span><span> |
Latest revision as of 04:04, 22 October 2021
CONTRIBUTIONS
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).