Difference between revisions of "Team:BIT-China/Project/Transduction"

 
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    <section class="content_container" id="mytop">
 
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          <h2 class="title-h2">Transduction</h2>
 
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            <h3 class="title-h3"></h3>
 
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            <p class="my-content-p">In order to enhance the signal transmission and amplification, we need to knock out some genes of yeast, including Ste2, Sst2 and Far1. Ste2, Sst2 and Far1 exist in endogenous GPCR pathways of yeast, which responds to the GPCR signal and start the downstream of the Signaling pathways. Ste2 interferes with the sweetness signals which respond to the pheromone. Knocking out Sst2 is necessary to remove the inhibition of sweetness signals. Far1 regulars the cell division which may have a bad influence on the yeast after the genes are knocked out. All in all, knocking out these genes is an essential step of the experiment. The yeast can cause the gene replacement or gene lost by homologous recombination, for that reason these genes will be knocked out by the means of designing the primers cleverly.</p>
 
  
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            <p class="my-content-p">To make our yeast detect sweeteners, we replace <span style= "font-style: oblique">ste2</span> receptor with Human sweet receptor T1R2-T1R3. So, first, we have to knock out <span style= "font-style: oblique"> ste2</span> gene. To decrease other interference factor we knock out <span style= "font-style: oblique"> sst2</span> gene and <span style= "font-style: oblique"> far1</span> gene. sst2 and far1 are genes that functioned to tunning signals among the MAPK pathway, they are believed to coordinated growth while yeast’s mitosis as described in the Design.</p>
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<section class="content_container" id="mytop">
  
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             <p class="my-content-p">By NCBI databases for obtaining <span style= "font-style: oblique"> ste2 </span><span style= "font-style: oblique"> sst2</span> and <span style= "font-style: oblique"> far1</span> gene sequence information, we find appropriate homologous arm in upstream and downstream genes. In order to knock out these genes effectively, we select different lengths of homologous arms, including 50bp, 200bp and 500bp. According to the wet-lab measurement, we find out 200bp and 500bp homologous arms are more efficient. </p>  
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        <h2 class="title-h2">Pathway optimization</h2>
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<div class="section-upline cd-section" id="knockout">
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             <h3 class="title-h3">gene knockout</h3>
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<p class="my-content-p">In order to amplify the intensity of the signal, we need to knock out some genes in yeast, including <i>ste2</i>, <i>sst2</i> and <i>far1</i> which exist in the endogenous mating pheromone response pathways in yeast and respond to the mating signal and transduction of downstream signal.</p>
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<div class="my-img-box" style="justify-content: flex-start;">
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            <table class="table-co">
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                <thead>
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                    <tr>
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                      <th style="width: 50%">Gene</th>
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                      <th>Function</th>
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                    </tr>
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                    <tbody>
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                    <tr>
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                        <td><i>ste2</i></td>
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                        <td>The GPCR of yeast pheromone receptor is Ste2 protein, which is an important factor during yeasts mating.</td>
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                    </tr>
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                    <tr>
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                        <td><i>sst2</i></td>
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                        <td>Sst2 protein is a phosphatase catalyzing GTP and producing GDP which will inhibit the release of G<sub>γ</sub>、<sub>β</sub> subunits. Gene knockout of <i>sst2</i> can improve the pheromone GPSTP.</td>
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                    </tr>
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                    <tr>
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                      <td><i>far1</i></td>
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                      <td>α factor is an active factor of the yeast pheromone GPSTP, which can cause the yeast cell cycle arresting in the G1 phase through Far1 protein, resulting in that cell growth is inhibited.</td>
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                    </tr>
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                </tbody>
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<p class="my-content-p">Gene knockout can be achieved by homologous recombination.</p>
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</div>
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<div class="section-upline cd-section" id="homologous">
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            <h3 class="title-h3">homologous recombination</h3>
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<p class="my-content-p">By analyzing <i>ste2</i>, <i>sst2</i> and <i>far1</i> gene sequences from the NCBI, we ensured the appropriate homologous area in the upstream and the downstream of these genes in <i>saccharomyces cerevisiae</i> genome. In order to knock out these genes effectively, we selected different lengths of homologous arms, 50bp, 200bp and 500bp. According to our experiment result, we found that 200bp and 500bp homologous arms worked more efficiently.</p>
  
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            <p class="my-content-p">We design 3 pairs of primers cleverly whose templates are the upstream homologous arm, marker and the downstream homologous arm respectively. The markers are Histone synthesis gene, Uracil synthesis gene and Trptophan synthesis gene, short termed His, Ura and Trp . His will replace <span style= "font-style: oblique"> sst2</span>, Ura will respanlace <span style= "font-style: oblique"> far1</span> and Trp will replace <span style= "font-style: oblique"> ste2</span>. Initially, we get 3 fragments by PCR and they have the overlap areas with each other. </p>
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<div class="my-content-box">
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            <p class="my-content-p">Secondly, the complete fragment observed by OE-PCR is converted to the yeast which has been already knocked out the endogenous gene Uracil, Histone and Trptophan. Additionally, the colony is chosen on the relevant nutritional deficiency medium, so that only the positive cloning can survival on it. </p>
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<img style="width: 60%; height: auto;" src="https://static.igem.org/mediawiki/2017/0/0d/T-BIT-China-2017e-1.png"/>
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<span>Fig.1 Homologous combination knock-out strategy</span>
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</div>
  
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            <p class="my-content-p">To verify whether the gene is actually knocked out and avoid the false positive colonies, we design the primer1,2,3 and 4 for each genes, as shown in the figure1. The primer 1 and primer 4 are on the yeast genome. The primer 2 is on the marker and primer 3 is on the gene which will be knocked out. </p>
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<p class="my-content-p">We designed 3 pairs of primers whose templates were the upstream homologous arm, the marker and the downstream homologous arm respectively. The markers were <i>Histone</i> synthesis gene, <i>Uracil</i> synthesis gene and <i>Tryptophan</i> synthesis gene, abbreviated as <i>his</i>, <i>ura</i> and <i>trp</i>. Initially, we got 3 fragments by PCR and they had their own overlapping areas with corresponding to each homologous arms. </p>
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                <img style="width: 60%; height: 60%" src="https://static.igem.org/mediawiki/2017/2/2c/T--BIT-China--2017project_transduction1.png" alt="">
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            <p class="my-content-p">If we get correct results by the primer 1 and 2 as well as nothing from primer 1 and 3, demonstrate that the gene is knocked out. Then we sequencing the PCR product using primer 1 and 4 to make sure the sequence is right.<a href="">click here to read results</a></p>
 
  
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            <p class="my-content-p">After knocking out far1, we test the growth curve of the yeast. Far1 protein is a cyclin-dependent kinase inhibitor. Deletion far1 gene can relieve inhibition. As you can see, it’s…… </p>
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<p class="my-content-p">Secondly, the recombination fragment obtained from OE-PCR was transformed to the <i>CEN.PK2-1C</i> which has been already knocked out the endogenous genes <i>ura, his</i> and <i>trp</i>. Additionally, the colony was chosen by the relevant nutritional deficiency medium. </p>
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            <p class="my-content-p">After knocking out sst2, we test the growth curve of the yeast. Sst2 protein is an important negative regulatory factor of GPSTP. When we knock out sst2 gene, the sensitivity of the yeast GPSTP could be improved. And the inhibition of cell growth will be enhanced.</p>
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<div class="section-upline cd-section" id="verification">
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                <img style="width: 60%; height: 60%" src="https://static.igem.org/mediawiki/2017/1/13/T--BIT-China--2017project_transduction2.png" alt="">
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            <h3 class="title-h3">verification</h3>
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            </div>
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<p class="my-content-p">To verify whether the gene was actually knocked out and to avoid false positive colonies, we designed primer1, 2, 3 and 4 for each gene, as shown in Fig.2. The primer 1 and primer 4 were designed according to the yeast genome. The primer 2 was designed according to the selection maker and primer 3 was to the gene which would be knocked out. </p>
  
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            <p class="my-content-p">As you can see, ΔSst2 strain is much more sensitive to α pheromone. Compared with CENPK2-1C strain, less amount of α pheromone can cause yeast growth arrest. </p>
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<div class="my-content-box">
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<img class="formula" src="https://static.igem.org/mediawiki/2017/f/f4/BIT-China_project-transduction_fig.1.png"/>
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<span>Fig.2 Verification strategy</span>
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<p class="my-content-p">We got positive result using the primer 1 and 2, meanwhile the negative result would be detected by using primer 1 and 3. Then we sequenced the PCR product using primer 1 and 4 to further confirm the realness of the results.</p>
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Latest revision as of 05:44, 31 October 2017

BIT-CHINA

Pathway optimization

gene knockout

In order to amplify the intensity of the signal, we need to knock out some genes in yeast, including ste2, sst2 and far1 which exist in the endogenous mating pheromone response pathways in yeast and respond to the mating signal and transduction of downstream signal.

Gene Function
ste2 The GPCR of yeast pheromone receptor is Ste2 protein, which is an important factor during yeasts mating.
sst2 Sst2 protein is a phosphatase catalyzing GTP and producing GDP which will inhibit the release of Gγ、β subunits. Gene knockout of sst2 can improve the pheromone GPSTP.
far1 α factor is an active factor of the yeast pheromone GPSTP, which can cause the yeast cell cycle arresting in the G1 phase through Far1 protein, resulting in that cell growth is inhibited.

Gene knockout can be achieved by homologous recombination.

homologous recombination

By analyzing ste2, sst2 and far1 gene sequences from the NCBI, we ensured the appropriate homologous area in the upstream and the downstream of these genes in saccharomyces cerevisiae genome. In order to knock out these genes effectively, we selected different lengths of homologous arms, 50bp, 200bp and 500bp. According to our experiment result, we found that 200bp and 500bp homologous arms worked more efficiently.

Fig.1 Homologous combination knock-out strategy

We designed 3 pairs of primers whose templates were the upstream homologous arm, the marker and the downstream homologous arm respectively. The markers were Histone synthesis gene, Uracil synthesis gene and Tryptophan synthesis gene, abbreviated as his, ura and trp. Initially, we got 3 fragments by PCR and they had their own overlapping areas with corresponding to each homologous arms.

Secondly, the recombination fragment obtained from OE-PCR was transformed to the CEN.PK2-1C which has been already knocked out the endogenous genes ura, his and trp. Additionally, the colony was chosen by the relevant nutritional deficiency medium.

verification

To verify whether the gene was actually knocked out and to avoid false positive colonies, we designed primer1, 2, 3 and 4 for each gene, as shown in Fig.2. The primer 1 and primer 4 were designed according to the yeast genome. The primer 2 was designed according to the selection maker and primer 3 was to the gene which would be knocked out.

Fig.2 Verification strategy

We got positive result using the primer 1 and 2, meanwhile the negative result would be detected by using primer 1 and 3. Then we sequenced the PCR product using primer 1 and 4 to further confirm the realness of the results.

TOP

Contact

Institute of Biotransformation and synthetic biosystem

School of life science

Beijing Institute of Technology

100081, Beijing

Email: lichun@bit.edu.cn