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

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      <section class="cd-section" id="Result">
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         <h2 class="title-h2">Result</h2>
 
         <h2 class="title-h2">Result</h2>
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        <div class="vs-content">
 
 
            
 
            
 
           <p class="my-content-p"> The strategy for the function of engineered CEN.PK2-1C  stains lacks <span class="my-oblique">△far1</span> +<span class="my-oblique">△sst2</span>, <span class="my-oblique">△sst</span> and <span class="my-oblique">△far1</span>+<span class="my-oblique">△sst2</span>+<span class="my-oblique">△ste2</span> were divided into the following parts; </p>
 
           <p class="my-content-p"> The strategy for the function of engineered CEN.PK2-1C  stains lacks <span class="my-oblique">△far1</span> +<span class="my-oblique">△sst2</span>, <span class="my-oblique">△sst</span> and <span class="my-oblique">△far1</span>+<span class="my-oblique">△sst2</span>+<span class="my-oblique">△ste2</span> were divided into the following parts; </p>
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           <div class="section-upline cd-section" id="Zone">
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           <h3 class="title-h3">Zone of inhibition</h3>
+
            <h3 class="title-h3">Zone of inhibition</h3>
−
          <h4 class="title-h4">The plan and method</h4>
+
            <h4 class="title-h4">The plan and method</h4>
−
          <p class="my-content-p">The growth of CEN.PK2-1C is inhibited by α factor,We want to  test the function of <span class="my-oblique">△sst2</span>, <span class="my-oblique">△far1</span>+<span class="my-oblique">△sst2</span> and <span class="my-oblique">△far1</span>+<span class="my-oblique">△sst2</span>+<span class="my-oblique">△ste2</span> by comparing with CEN.PK2-1C .</p>
+
            <p class="my-content-p">The growth of CEN.PK2-1C is inhibited by α factor,We want to  test the function of <span class="my-oblique">△sst2</span>, <span class="my-oblique">△far1</span>+<span class="my-oblique">△sst2</span> and <span class="my-oblique">△far1</span>+<span class="my-oblique">△sst2</span>+<span class="my-oblique">△ste2</span> by comparing with CEN.PK2-1C .</p>
−
          <p class="my-content-p">(1)The influence of the growth of bacteria caused by different amount of α factor in the solid plate.</p>
+
            <p class="my-content-p">(1)The influence of the growth of bacteria caused by different amount of α factor in the solid plate.</p>
−
          <p class="my-content-p">(2)The observed difference among the function of engineered CEN.PK2-1C strains, <span class="my-oblique">△far1</span>+<span class="my-oblique">△sst2</span>, <span class="my-oblique">△far1</span>+<span class="my-oblique">△sst2</span>+<span class="my-oblique">△ste2</span>  acted on the growth of Cen.PK2-1C, <span class="my-oblique">△sst2</span>, <span class="my-oblique">△far1</span>+<span class="my-oblique">△sst2</span>, <span class="my-oblique">△far1</span>+<span class="my-oblique">△sst2</span>+<span class="my-oblique">△ste2</span> impaired  by different amount of α factor.</p>
+
            <p class="my-content-p">(2)The observed difference among the function of engineered CEN.PK2-1C strains, <span class="my-oblique">△far1</span>+<span class="my-oblique">△sst2</span>, <span class="my-oblique">△far1</span>+<span class="my-oblique">△sst2</span>+<span class="my-oblique">△ste2</span>  acted on the growth of Cen.PK2-1C, <span class="my-oblique">△sst2</span>, <span class="my-oblique">△far1</span>+<span class="my-oblique">△sst2</span>, <span class="my-oblique">△far1</span>+<span class="my-oblique">△sst2</span>+<span class="my-oblique">△ste2</span> impaired  by different amount of α factor.</p>
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          <h4 class="title-h4">The results and discussion</h4>
+
            <h4 class="title-h4">The results and discussion</h4>
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          <p class="my-content-p">(1)The bacteria strain of CEN.PK2-1C stops growing in G1 due to the effects acted by the α factor and Zone of inhibition appears in the panel.</p>
+
            <p class="my-content-p">(1)The bacteria strain of CEN.PK2-1C stops growing in G1 due to the effects acted by the α factor and Zone of inhibition appears in the panel.</p>
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          <div class="my-content-box">
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            <div class="my-content-box">
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            <img class="formula" style="width: 40%; height: 40%" src="https://static.igem.org/mediawiki/2017/4/46/T-BIT-China-2017result-1.png">
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              <img class="formula" style="width: 40%; height: 40%" src="https://static.igem.org/mediawiki/2017/4/46/T-BIT-China-2017result-1.png">
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            <span>Fig.1 With the increase of the concentration of the alpha factor,there is no inhibition zone.</span>
+
              <span>Fig.1 With the increase of the concentration of the alpha factor,there is no inhibition zone.</span>
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          </div>
+
−
          <p class="my-content-p">(2)<span class="my-oblique">△sst2</span> strain (sst2 is knocked out successfully) and the sensitivity of <span class="my-oblique">△sst2</span> strain to α factor will be increased. Meanwhile the larger Zone of inhibition will appear when the amount of α factor is reduced.</p>
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          <div class="my-img-box2">
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            <div class="my-img-left">
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              <img style="width: 62%; height: 62%; margin-right:-60px" src="https://static.igem.org/mediawiki/2017/9/94/T-BIT-China-2017result-2.png">
+
 
             </div>
 
             </div>
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             <div class="my-img-right">
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            <p class="my-content-p">(2)<span class="my-oblique">△sst2</span> strain (sst2 is knocked out successfully) and the sensitivity of <span class="my-oblique">△sst2</span> strain to α factor will be increased. Meanwhile the larger Zone of inhibition will appear when the amount of α factor is reduced.</p>
−
               <img  style="width: 59%; height: 59%" src="https://static.igem.org/mediawiki/2017/a/a8/T-BIT-China-2017result-3.png">
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             <div class="my-img-box2">
 +
              <div class="my-img-left">
 +
                <img style="width: 62%; height: 62%; margin-right:-60px" src="https://static.igem.org/mediawiki/2017/9/94/T-BIT-China-2017result-2.png">
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               </div>
 +
              <div class="my-img-right">
 +
                <img  style="width: 59%; height: 59%" src="https://static.igem.org/mediawiki/2017/a/a8/T-BIT-China-2017result-3.png">
 +
              </div>
 
             </div>
 
             </div>
−
          </div>
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            <div class="my-content-box2">
−
          <div class="my-content-box2">
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              <span>Fig.2 With the increase of the concentration of the alpha factor,the radius of zone of inhibition increases.</span>
−
            <span>Fig.2 With the increase of the concentration of the alpha factor,the radius of zone of inhibition increases.</span>
+
−
          </div>
+
−
          <p class="my-content-p">(3)The far1 of <span class="my-oblique">△far1</span>+<span class="my-oblique">△sst2</span>, <span class="my-oblique">△far1</span>+<span class="my-oblique">△sst2</span>+<span class="my-oblique">△ste2</span> is knocked out successfully, and this strain will still keep growing under the effects acted by α factor and Zone of inhibition will not be shown either.</p>
+
−
          <div class="my-img-box2">
+
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            <div class="my-img-left">
+
−
              <img style="width: 50%; height: 50%" src="https://static.igem.org/mediawiki/2017/c/c5/T-BIT-China-2017result-4.png">
+
 
             </div>
 
             </div>
−
             <div class="my-img-right">
+
            <p class="my-content-p">(3)The far1 of <span class="my-oblique">△far1</span>+<span class="my-oblique">△sst2</span>, <span class="my-oblique">△far1</span>+<span class="my-oblique">△sst2</span>+<span class="my-oblique">△ste2</span> is knocked out successfully, and this strain will still keep growing under the effects acted by α factor and Zone of inhibition will not be shown either.</p>
−
               <img style="width: 54%; height: 54%" src="https://static.igem.org/mediawiki/2017/6/6d/T-BIT-China-2017result-5.png">
+
             <div class="my-img-box2">
 +
               <div class="my-img-left">
 +
                <img style="width: 50%; height: 50%" src="https://static.igem.org/mediawiki/2017/c/c5/T-BIT-China-2017result-4.png">
 +
              </div>
 +
              <div class="my-img-right">
 +
                <img style="width: 54%; height: 54%" src="https://static.igem.org/mediawiki/2017/6/6d/T-BIT-China-2017result-5.png">
 +
              </div>
 
             </div>
 
             </div>
 +
            <div class="my-content-box2">
 +
              <span>Fig.3 With the increase of the concentration of the alpha factor,Δfar1+Δsst2 and Δfar1+Δsst2+Δste2 of CEN.PK2-1C is no inhibition zone.</span>
 +
            </div>
 +
           
 +
            <p class="my-content-p">Conclusion： Because far1 can make cellular period stop at the G1 stage by  α factor and sst2 can reduce the intensity of the transmitted signal. So we want to knock out of  far1 and  sst2.From the above results we can see that in the α factor concentration range, the strain will be inhibited by  α factor. With the increase of the concentration of the alpha factor,the radius of Zone of inhibition increases and the inhibitory effect of the growth of the strain was enhanced. And more obvious. Knocking out of  the sst2 gene, making the strain respond to α factor enhanced.</p>
 
           </div>
 
           </div>
−
          <div class="my-content-box2">
 
−
            <span>Fig.3 With the increase of the concentration of the alpha factor,Δfar1+Δsst2 and Δfar1+Δsst2+Δste2 of CEN.PK2-1C is no inhibition zone.</span>
 
−
          </div>
 
−
         
 
−
          <p class="my-content-p">Conclusion： Because far1 can make cellular period stop at the G1 stage by  α factor and sst2 can reduce the intensity of the transmitted signal. So we want to knock out of  far1 and  sst2.From the above results we can see that in the α factor concentration range, the strain will be inhibited by  α factor. With the increase of the concentration of the alpha factor,the radius of Zone of inhibition increases and the inhibitory effect of the growth of the strain was enhanced. And more obvious. Knocking out of  the sst2 gene, making the strain respond to α factor enhanced.</p>
 
  
 +
          <div class="section-upline cd-section" id="curve">
 +
            <h3 class="title-h3">The growth curve</h3>
 +
            <p class="my-content-p"> The impacts acted by the various concentration of α factor to the growth of the bacteria immersed into liquid.</p>
 +
            <p class="my-content-p"> The research of the impacts acted by various concentration of α factor to the growth of Cen.PK2-1C,  <span class="my-oblique">△sst2</span> of CEN.PK2-1C.</p>
 +
            <h4 class="title-h4">The plan and method</h4>
 +
            <p class="my-content-p">The gowth of engineered CEN.PK2-1C strains were determined by measuring the initial rates of α factor at concentrations of 0-15 µM . We take out 5ml cultivating bacteria liquid of each of them during  30h and correct them by the YPD solution which not inoculates yeasts after  shake culture. We obtain the value of OD600 at position of wavelength of  600nm by UV-Vis spectrophotometer.</p>
 +
            <h4 class="title-h4">The results and discuss</h4>
 +
           
 +
            <p class="my-content-p">(1)With the increasing  concentration of α factor, the growth curve of CEN.PK2-1C is inhibited by α factor.</p>
 +
            <div class="my-content-box">
 +
              <img class="formula" src="https://static.igem.org/mediawiki/2017/e/e3/T-BIT-China-2017result-6.png"/>
 +
              <span>Fig.4 With the increasing  concentration of α factor, the growth curve of CEN.PK2-1C is inhibited by α factor.</span>
 +
            </div>
  
 +
            <p class="my-content-p">(2)the growth curve of <span class="my-oblique">△sst2</span>  is bit lower than CEN.PK2-1C. It’s strongly inhibited by α factor.</p>
 +
            <div class="my-content-box">
 +
              <img class="formula" src="https://static.igem.org/mediawiki/2017/4/45/T-BIT-China-2017result-7.png"/>
 +
              <span>Fig.5 With the increasing  concentration of α factor, the growth curve of Δsst2  is inhibited by α factor.</span>
 +
            </div>
  
 +
            <p class="my-content-p">Conclusion：From the above results we can see that in the α factor concentration range, the strain will be inhibited by  α factor. With the increase of the concentration of the alpha factor, the inhibitory effect of the growth of the strain was enhanced. And more obvious. Knocking down the sst2 gene, making the strain respond to α factor enhanced.</p>
 +
          </div>
  
 +
          <div class="section-upline cd-section" id="Fluorescence">
 +
            <h3 class="title-h3">Fluorescence</h3>
 +
            <p class="my-content-p">Test the expression of Pfus+mRFP+cyc1t+pRS42K in engineered CEN.PK2-1C strains, <span class="my-oblique">△far1</span>+<span class="my-oblique">△sst2</span>, <span class="my-oblique">△far1</span>+<span class="my-oblique">△sst2</span>+<span class="my-oblique">△ste2</span>, <span class="my-oblique">△sst2</span>.</p>
 +
            <p class="my-content-p">We research the impacts acted by various concentration of α factor to the amount of expression of fluorescence  as well as the the growth of engineered CEN.PK2-1C strains, <span class="my-oblique">△far1</span>+<span class="my-oblique">△sst2、△far1</span>+<span class="my-oblique">△sst2</span>+<span class="my-oblique">△ste2，△sst2</span> containing the examined circuit.</p>
 +
            <h4 class="title-h4">The plan and method</h4>
 +
            <p class="my-content-p">We set the concentration of alpha factor as 0umol/L，1 umol/L， 2.5 umol/L， 5 umol/L，10 umol/L，15 umol/L.We take out 5ml cultivating bacteria liquid of each of them after 0、4、8、10、12、14、16、18、20、22、24、26、28、30h respectively and correct them by the YPD solution which not inoculated yeasts after shake culture. Afterwards,the intensity of triggered fluorescence is measured in microplate reader(584/607).We create the coordianate system of the expression of fluorescence of engeneering yeast by setting the value of that intensity of triggered fluorescence/OD600 as Y-axis and time taken as X- axis.</p>
 +
            <h4 class="title-h4">The results and discuss</h4>
 +
            <div class="my-content-box">
 +
              <img class="formula" src="https://static.igem.org/mediawiki/2017/0/00/T-BIT-China-2017result-8.png" />
 +
              <span>Fig.6 With the increase of the concentration of α factor, the fluorescence intensity of CEN.PK2-1C increased.</span>
 +
            </div>
 +
            <div class="my-content-box">
 +
              <img class="formula" src="https://static.igem.org/mediawiki/2017/f/f6/T-BIT-China-2017result-9.png" />
 +
              <span>Fig.7 With the increase of the concentration of α factor, the fluorescence intensity of CEN.PK2-1C increased.</span>
 +
            </div>
 +
            <div class="my-content-box">
 +
              <img class="formula" src="https://static.igem.org/mediawiki/2017/0/08/T-BIT-China-2017result-10.png" />
 +
              <span>Fig.8 The strongest expression of fluorescence belongs to <span class="my-oblique">△far1</span>+<span class="my-oblique">△sst2</span>, while that of <span class="my-oblique">△far1</span>+<span class="my-oblique">△sst2</span>+<span class="my-oblique">△ste2</span> and CEN.PK2-1C are similar.</span>
 +
            </div>
  
 +
            <p class="my-content-p">Conclusion：From the above results we can see that in the α factor concentration range, the expression of mRFP was induced. With the increase of the concentration of the alpha factor, the fluorescence expression was enhanced. And more obvious. Knocking down the sst2 and far1gene, making the strain respond to α factor enhanced.</p>
 +
            </div>
  
  
−
          <h3 class="title-h3">The growth curve </h3>
+
      <div class="article-nav">
−
          <p class="my-content-p"> The impacts acted by the various concentration of α factor to the growth of the bacteria immersed into liquid.
+
        <a href="https://2017.igem.org/Team:BIT-China/Project/Detection" class="article-nav-left">
−
           <p class="my-content-p"> The research of the impacts acted by various concentration of α factor to the growth of Cen.PK2-1C,  <span class="my-oblique">△sst2</span> of CEN.PK2-1C.
+
           <img class="article-caption" src="https://static.igem.org/mediawiki/2017/a/a4/T--BIT-China--2017previous.png" alt="">  
−
          <h4 class="title-h4">The plan and method</h4>
+
        </a>
−
          <p class="my-content-p">The gowth of engineered CEN.PK2-1C strains were determined by measuring the initial rates of α factor at concentrations of 0-15 µM . We take out 5ml cultivating bacteria liquid of each of them during  30h and correct them by the YPD solution which not inoculates yeasts after  shake culture. We obtain the value of OD600 at position of wavelength of  600nm by UV-Vis spectrophotometer.</p>
+
      </div>
−
          <h4 class="title-h4">The results and discuss</h4>
+
    </section>
−
         
+
    <nav id="cd-vertical-nav">
−
          <p class="my-content-p">(1)With the increasing  concentration of α factor, the growth curve of CEN.PK2-1C is inhibited by α factor.</p>
+
    <ul>
−
          <div class="my-content-box">
+
      <li>
−
            <img class="formula" src="https://static.igem.org/mediawiki/2017/e/e3/T-BIT-China-2017result-6.png"/>
+
        <a href="#Zone" data-number="1">
−
            <span>Fig.4 With the increasing  concentration of α factor, the growth curve of CEN.PK2-1C is inhibited by α factor.</span>
+
        <span class="cd-dot"></span>
−
          </div>
+
        <span class="cd-label">Zone of inhibition</span>
 +
        </a>
 +
      </li>
  
−
          <p class="my-content-p">(2)the growth curve of <span class="my-oblique">△sst2</span>  is bit lower than CEN.PK2-1C. It’s strongly inhibited by α factor.</p>
+
      <li>
−
          <div class="my-content-box">
+
        <a href="#curve" data-number="2">
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            <img class="formula" src="https://static.igem.org/mediawiki/2017/4/45/T-BIT-China-2017result-7.png"/>
+
        <span class="cd-dot"></span>
−
            <span>Fig.5 With the increasing  concentration of α factor, the growth curve of Δsst2  is inhibited by α factor.</span>
+
        <span class="cd-label">The growth curve</span>
−
          </div>
+
        </a>
 +
      </li>
  
−
          <p class="my-content-p">Conclusion：From the above results we can see that in the α factor concentration range, the strain will be inhibited by  α factor. With the increase of the concentration of the alpha factor, the inhibitory effect of the growth of the strain was enhanced. And more obvious. Knocking down the sst2 gene, making the strain respond to α factor enhanced.</p>
+
      <li>
 +
        <a href="#Fluorescence" data-number="3">
 +
        <span class="cd-dot"></span>
 +
        <span class="cd-label">Fluorescence</span>
 +
        </a>
 +
      </li>
  
−
 
+
   
−
 
+
    </ul>
−
          <h3 class="title-h3">Fluorescence</h3>
+
  </nav>
−
          <p class="my-content-p">Test the expression of Pfus+mRFP+cyc1t+pRS42K in engineered CEN.PK2-1C strains, <span class="my-oblique">△far1</span>+<span class="my-oblique">△sst2</span>, <span class="my-oblique">△far1</span>+<span class="my-oblique">△sst2</span>+<span class="my-oblique">△ste2</span>, <span class="my-oblique">△sst2</span>.</p>
+
<a class="my-backTop" href="#mytop">
−
          <p class="my-content-p">We research the impacts acted by various concentration of α factor to the amount of expression of fluorescence  as well as the the growth of engineered CEN.PK2-1C strains, <span class="my-oblique">△far1</span>+<span class="my-oblique">△sst2、△far1</span>+<span class="my-oblique">△sst2</span>+<span class="my-oblique">△ste2，△sst2</span> containing the examined circuit.</p>
+
      <span>TOP</span>
−
          <h4 class="title-h4">The plan and method</h4>
+
      <img src="https://static.igem.org/mediawiki/2017/f/fd/Bit-china-2017backTop.png" alt="">  
−
          <p class="my-content-p">We set the concentration of alpha factor as 0umol/L，1 umol/L， 2.5 umol/L， 5 umol/L，10 umol/L，15 umol/L.We take out 5ml cultivating bacteria liquid of each of them after 0、4、8、10、12、14、16、18、20、22、24、26、28、30h respectively and correct them by the YPD solution which not inoculated yeasts after shake culture. Afterwards,the intensity of triggered fluorescence is measured in microplate reader(584/607).We create the coordianate system of the expression of fluorescence of engeneering yeast by setting the value of that intensity of triggered fluorescence/OD600 as Y-axis and time taken as X- axis.</p>
+
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          <h4 class="title-h4">The results and discuss</h4>
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            <span>Fig.6 With the increase of the concentration of α factor, the fluorescence intensity of CEN.PK2-1C increased.</span>
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            <span>Fig.7 With the increase of the concentration of α factor, the fluorescence intensity of CEN.PK2-1C increased.</span>
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            <span>Fig.8 The strongest expression of fluorescence belongs to <span class="my-oblique">△far1</span>+<span class="my-oblique">△sst2</span>, while that of <span class="my-oblique">△far1</span>+<span class="my-oblique">△sst2</span>+<span class="my-oblique">△ste2</span> and CEN.PK2-1C are similar.</span>
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          <p class="my-content-p">Conclusion：From the above results we can see that in the α factor concentration range, the expression of mRFP was induced. With the increase of the concentration of the alpha factor, the fluorescence expression was enhanced. And more obvious. Knocking down the sst2 and far1gene, making the strain respond to α factor enhanced.</p>
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Revision as of 12:31, 24 October 2017

BIT-CHINA

Result

The strategy for the function of engineered CEN.PK2-1C stains lacks △far1 +△sst2, △sst and △far1+△sst2+△ste2 were divided into the following parts;

(1). Testing the inhibitive effects on the growth of engineered CEN.PK2-1C, whose cellular period can be stopped at the G1 stage by α factor.

(2). Expression and identification of Pfus+RFP+cyc1t+PRS42K in engineered CEN.PK2-1C strains △far1+△sst2, △far1+△sst2+△ste2.

Zone of inhibition

The plan and method

The growth of CEN.PK2-1C is inhibited by α factor,We want to test the function of △sst2, △far1+△sst2 and △far1+△sst2+△ste2 by comparing with CEN.PK2-1C .

(1)The influence of the growth of bacteria caused by different amount of α factor in the solid plate.

(2)The observed difference among the function of engineered CEN.PK2-1C strains, △far1+△sst2, △far1+△sst2+△ste2 acted on the growth of Cen.PK2-1C, △sst2, △far1+△sst2, △far1+△sst2+△ste2 impaired by different amount of α factor.

The results and discussion

(1)The bacteria strain of CEN.PK2-1C stops growing in G1 due to the effects acted by the α factor and Zone of inhibition appears in the panel.

Fig.1 With the increase of the concentration of the alpha factor,there is no inhibition zone.

(2)△sst2 strain (sst2 is knocked out successfully) and the sensitivity of △sst2 strain to α factor will be increased. Meanwhile the larger Zone of inhibition will appear when the amount of α factor is reduced.

Fig.2 With the increase of the concentration of the alpha factor,the radius of zone of inhibition increases.

(3)The far1 of △far1+△sst2, △far1+△sst2+△ste2 is knocked out successfully, and this strain will still keep growing under the effects acted by α factor and Zone of inhibition will not be shown either.

Fig.3 With the increase of the concentration of the alpha factor,Δfar1+Δsst2 and Δfar1+Δsst2+Δste2 of CEN.PK2-1C is no inhibition zone.

Conclusion： Because far1 can make cellular period stop at the G1 stage by α factor and sst2 can reduce the intensity of the transmitted signal. So we want to knock out of far1 and sst2.From the above results we can see that in the α factor concentration range, the strain will be inhibited by α factor. With the increase of the concentration of the alpha factor,the radius of Zone of inhibition increases and the inhibitory effect of the growth of the strain was enhanced. And more obvious. Knocking out of the sst2 gene, making the strain respond to α factor enhanced.

The growth curve

The impacts acted by the various concentration of α factor to the growth of the bacteria immersed into liquid.

The research of the impacts acted by various concentration of α factor to the growth of Cen.PK2-1C, △sst2 of CEN.PK2-1C.

The plan and method

The gowth of engineered CEN.PK2-1C strains were determined by measuring the initial rates of α factor at concentrations of 0-15 µM . We take out 5ml cultivating bacteria liquid of each of them during 30h and correct them by the YPD solution which not inoculates yeasts after shake culture. We obtain the value of OD600 at position of wavelength of 600nm by UV-Vis spectrophotometer.

The results and discuss

(1)With the increasing concentration of α factor, the growth curve of CEN.PK2-1C is inhibited by α factor.

Fig.4 With the increasing concentration of α factor, the growth curve of CEN.PK2-1C is inhibited by α factor.

(2)the growth curve of △sst2 is bit lower than CEN.PK2-1C. It’s strongly inhibited by α factor.

Fig.5 With the increasing concentration of α factor, the growth curve of Δsst2 is inhibited by α factor.

Conclusion：From the above results we can see that in the α factor concentration range, the strain will be inhibited by α factor. With the increase of the concentration of the alpha factor, the inhibitory effect of the growth of the strain was enhanced. And more obvious. Knocking down the sst2 gene, making the strain respond to α factor enhanced.

Fluorescence

Test the expression of Pfus+mRFP+cyc1t+pRS42K in engineered CEN.PK2-1C strains, △far1+△sst2, △far1+△sst2+△ste2, △sst2.

We research the impacts acted by various concentration of α factor to the amount of expression of fluorescence as well as the the growth of engineered CEN.PK2-1C strains, △far1+△sst2、△far1+△sst2+△ste2，△sst2 containing the examined circuit.

The plan and method

We set the concentration of alpha factor as 0umol/L，1 umol/L， 2.5 umol/L， 5 umol/L，10 umol/L，15 umol/L.We take out 5ml cultivating bacteria liquid of each of them after 0、4、8、10、12、14、16、18、20、22、24、26、28、30h respectively and correct them by the YPD solution which not inoculated yeasts after shake culture. Afterwards,the intensity of triggered fluorescence is measured in microplate reader(584/607).We create the coordianate system of the expression of fluorescence of engeneering yeast by setting the value of that intensity of triggered fluorescence/OD600 as Y-axis and time taken as X- axis.

The results and discuss

Fig.6 With the increase of the concentration of α factor, the fluorescence intensity of CEN.PK2-1C increased.
Fig.7 With the increase of the concentration of α factor, the fluorescence intensity of CEN.PK2-1C increased.
Fig.8 The strongest expression of fluorescence belongs to △far1+△sst2, while that of △far1+△sst2+△ste2 and CEN.PK2-1C are similar.

Conclusion：From the above results we can see that in the α factor concentration range, the expression of mRFP was induced. With the increase of the concentration of the alpha factor, the fluorescence expression was enhanced. And more obvious. Knocking down the sst2 and far1gene, making the strain respond to α factor enhanced.

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Contact

Institute of Biotransformation and synthetic biosystem

School of life science

Beijing Institute of Technology

100081, Beijing

Email: lichun@bit.edu.cn