Difference between revisions of "Team:Peking/Improve"

 
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                     <h1>Demonstrate</h1>
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                     <h1>Improvement</h1>
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                    <p class="title1" style="text-align:center">In this section, you could see the demonstration.</p>
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                                     <h4><a href="javascript:void(0);" onclick="naver('A')">Overview</a></h4>
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                                     <h4><a href="javascript:void(0);" onclick="naver('A')">&bull;Fused&nbsp;with&nbsp;yEGFP</a></h4>
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                                    <h4><a href="javascript:void(0);" onclick="naver('B')">Phase&nbsp;Separation</a></h4>
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                                     <h4><a href="javascript:void(0);" onclick="naver('B')">&bull;Phase&nbsp;Separation</a></h4>
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                                    <ul>
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                                        <li><a href="javascript:void(0);" onclick="naver('B1')">Spontaneous</a></li>
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                                        <li><a href="javascript:void(0);" onclick="naver('B2')">The&nbsp;formation</a></li>
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                                     <h4><a href="javascript:void(0);" onclick="naver('C')">Functional&nbsp;Organelles</a></h4>
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                                    <h4><a href="javascript:void(0);" onclick="naver('D')">Perspective</a></h4>
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                             <div class="texttitle">Fuse with yEGFP
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                             <div class="texttitle">Fused with yEGFP
 
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                                     <p>We fuse part <a href="http://parts.igem.org/Part:BBa_K209496">BBa_K209496</a> (Frb) and <a href="http://parts.igem.org/Part:BBa_K209496">BBa_K209023</a> (FKBP) with yEGFP, which make it visible in the yeast under fluorescent microscope. Then we got part<a href="http://parts.igem.org/Part:BBa_K2601008">BBa_K2601008(FKBP-yEGFP)</a> and <a href="http://parts.igem.org/Part:BBa_K2601007">BBa_K2601007(Frb-yEGFP).</a> </p>
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                                     <p>We fused part Frb<a href="http://parts.igem.org/Part:BBa_K209496"> (BBa_K209496) </a> and FKBP<a href="http://parts.igem.org/Part:BBa_K209023"> (BBa_K209023) </a>with yEGFP, which made it visible in the yeast under fluorescent microscope. Then we got part Frb-yEGFP<a href="http://parts.igem.org/Part:BBa_K2601007"> (BBa_K2601007) </a> and FKBP-yEGFP<a href="http://parts.igem.org/Part:BBa_K2601008"> (BBa_K2601008) </a>.</p>
 
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                                     <p>Afterward, we drive the expression of FKBP-yEGFP and Frb-yEGFP with 4 promoters pUra3, pTet07,pTEF1 and PDH3. The expression of these promoters were measure by flow cytometry(Fig. 1). We then got prats <a href="http://parts.igem.org/Part:BBa_K2601021">BBa_K2601021 (Tet07-Frb-yEGFP)</a>, <a href="http://parts.igem.org/Part:BBa_K2601023"> K2601023 (PDH3-Frb-yEGFP)</a>, <a href="http://parts.igem.org/Part:BBa_K2601025">BBa_K2601025 (Tet07-FKBP-yEGFP)</a>, <a href="http://parts.igem.org/Part:BBa_K2601026">BBa_K2601026 (TEF1-FKBP-yEGFP)</a>, <a href="http://parts.igem.org/Part:BBa_K2601027">K2601027 (PDH3-FKBP-yEGFP)</a>. </p>
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                                     <p>Then we characterized the expression of Frb-yEGFP and FKBP-yEGFP with 4 promoters pUra3, pTet07, pTEF1 and PDH3. The expression of these promoters was measured through flow cytometry (Fig. 1).  
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<br/>
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We made these improved prats:
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<br/>
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Tet07-Frb-yEGFP<a href="http://parts.igem.org/Part:BBa_K2601021"> (BBa_K2601021) </a>;
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<br/>
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PDH3-Frb-yEGFP<a href="http://parts.igem.org/Part:BBa_K2601023"> (BBa_K2601023) </a>;
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<br/>
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Tet07-FKBP-yEGFP<a href="http://parts.igem.org/Part:BBa_K2601025"> (BBa_K2601025) </a>;
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<br/>
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TEF1-FKBP-yEGFP<a href="http://parts.igem.org/Part:BBa_K2601026"> (BBa_K2601026) </a>;
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<br/>
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PDH3-FKBP-yEGFP<a href="http://parts.igem.org/Part:BBa_K2601027"> (BBa_K2601027) </a>.
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</p>
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<br/>
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<img src="https://static.igem.org/mediawiki/parts/f/f7/T--Peking--promoter-strength-new.png">
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<p>Figure. 1 The strength of three different yeast promoters tested through flow cytometry.</p>
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<div class="texttitle">Phase Separation System
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          <div class="texttitle">Phase separation system
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                                    <div class="ordi">1.</div>
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                                    <h3>Spontaneous and induced synthetic organelles can be formed by phase separation</h3>
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                                    <p>Moreover, we fused Frb-yEGFP with HOtag6 and FKBP-yEGFP with HOtag3. These parts can form phase separation in the presence of rapamycin (Fig. 2). The original part Frb<a href="http://parts.igem.org/Part:BBa_K209496"> (BBa_K209496) </a>and FKBP<a href="http://parts.igem.org/Part:BBa_K209023"> (BBa_K209023) </a>doesn't have this function.
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We uploaded 2 parts:
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                            <div class="coll">
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Frb-yEGFP-HOTag6<a href="http://parts.igem.org/Part:BBa_K2601010"> (BBa_K2601010) </a>;
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FKBP-yEGFP-HOTag3<a href="http://parts.igem.org/Part:BBa_K2601011"> (BBa_K2601011) </a>.
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</p>
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<img src="https://static.igem.org/mediawiki/2018/9/97/T--Peking--Demof4.png">
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<figcaption style="text-align:justify; text-justify:inter-ideograph;">
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Figure. 2  The improvement of Frb and FKBP fused with yEGFP.
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Figure. 2A The structure of Frb and FKBP. Rapamycin induces the interaction between them.
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Figure. 2B Design of RapaSPOT. FKBP is fused with mCherry and HOTag3 while Frb is fused with yEGFP and HOTag6. After adding rapamycin, they are expected self-organizing to form large assemblies, which will be organelles in cells.
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Figure. 2C Granules formed in chemical-induced SPOT after adding rapamycin. Ura3-FKBP-HOTag3 with mCherry and PDH3-Frb-HOTag6 with yEGFP are transferred and expressed in S. cerevisiae. Fluorescence images in both GFP and mCherry channels of cells are taken after adding 10 μM Figure. 2D Growth curves of wild-type yeast (black curve) and yeast strains of Ura3-FKBP-HOTag3 and PDH3-Frb-HOTag6 (red curve). They are measured after adding different concentration of rapamycin for 24 hours. Wild-type yeast cannot grow and reproduce normally while recombination yeast strains of FKBP-HOTag3 and Frb-HOTag6 can survive when exposed to rapamycin.
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<br/><br/><br/>
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<p>
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Then we characterized the expression of Frb-yEGFP-HOTag6 with 3 promoters pUra3, pTEF1 and PDH3. As our <a href="https://2018.igem.org/Team:Peking/Model">Modeling</a> work predicts, the kinetics of a system depends on the concentration of the components and the interaction strength(Fig .3). We successfully uploaded 5 parts form them:
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pTet07-Frb-yEGFP-HOTag6<a href="http://parts.igem.org/Part:BBa_K2601032"> (BBa_K2601032) </a>;
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<br/>
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pTEF1-Frb-yEGFP-HOTag6<a href="http://parts.igem.org/Part:BBa_K2601033"> (BBa_K2601033) </a>;
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PDH3-Frb-yEGFP-HOTag6<a href="http://parts.igem.org/Part:BBa_K2601034"> (BBa_K2601034) </a>;
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<br/>
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pTEF1-FKBP-yEGFP-HOTag3<a href="http://parts.igem.org/Part:BBa_K2601011"> (BBa_K2601011) </a>;
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<br/>
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PDH3-yEGFP-HOTag3<a href="http://parts.igem.org/Part:BBa_K2601011"> (BBa_K2601011) </a>.
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</p>
  
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                                    <p>What's more, we fused Frb-yEGFP with HOtag6 and fused FKBP-yEGFP with HOtag3. These two parts can form phase separation in the presence of rapamycin. The original part <a href="http://parts.igem.org/Part:BBa_K209496">BBa_K209496</a> (Frb) and <a href="http://parts.igem.org/Part:BBa_K209496">BBa_K209023</a> (FKBP) has not this function.</p>
 
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                                    <p>We used SUMO-SIM interaction module to build a spontaneous organelle. When two components are expressed in yeasts, granules with the two fluorescence proteins can be observed in vivo (Figure1.b). </p>
 
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                                    <p>Meanwhile, by rapamycin induced interaction module, FKBP-Frb, we have built an inducible organelle. We can see granules occurs in yeasts within minutes after adding the inducer.</a> </p>
 
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Figure1.a The basic design of synthetic organelles with florescence reporters. <img src="https://static.igem.org/mediawiki/2018/3/36/T--Peking--Logo.png" style="width:100%;" alt="">（这里可能需要一张cartoon的设计图）
 
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            b, c fluorescence images of spontaneous organelles (SUMO-SIM based) and inducible synthetic organelles (FKBP-Frb based, after adding 10000 nM rapamycin)<br/><br/>
 
  
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<img src="https://static.igem.org/mediawiki/2018/d/d8/T--Peking--Demof5.png"><br/>
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<figcaption style="text-align:justify; text-justify:inter-ideograph;">
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Figure. 3  The improvement of Frb and FKBP fused with yEGFP regulated by four kinds of promoters.  
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Figure. 3A The formation of SPOT can be described as a phase separation process of three components. Two important variables, the concentration of components and the interaction strength are marked in the figure.
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                                    <h3>The formation of organelles has flexible but predictable properties and kinetics in different conditions</h3>
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Figure. 3B Flow cytometry results of three promoters (pUra3, pTEF2, and PDH3). The expression level of Ura3 is the lowest while PDH3 is the strongest promoter.
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Figure. 3C RapaSPOT of different promoter combinations after 10 μM rapamycin induction. Two axes stand for the expression level of components. After 3 hours, only SPOT system with high level of Frb can be observed.
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Figure. 3D Proportion of yeast with granules after rapamycin induction. The rapamycin concentration range is from 1μM to 100 μM.
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                                    <p>Then we combined <a href="https://2018.igem.org/Team:Peking/Phase_Separation_M"/>modeling of phase separation</a> and experiment to research the kinetics of the organelles formation process expecting that a well-characterized system can reach its whole potential in complex applications. </p>
 
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                                    <p>As the model predicts, the concentration of components and the interaction strength affect the kinetics of phase separation. First we controlled the expression levels of components by using several stable or inducible promoters and observe the system's behavior. We found that the formation of organelles happened in specific promoter combinations and can be controlled by inducible promoters. The analysis result does not only fit well with the simulation, but provides potential methods to control the organelles in applications. </p>
 
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Figure2 (a) Phase diagram of a phase separation system with three components(simulation). To fit our system, the x-axis and the y-axis stands for the two components in the granules. The asymmetry comes from the assumption that the two components have different interactions with water.
 
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(b) Fluorescence movies of different promoter combinations of FKBP-Frb mediated system after adding rapamycin. Only in specific combinations, synthetic organelles can be formed by phase separation.
 
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(c) The formation process of SUMO-SIM mediated synthetic organelles can be controlled by inducible promoters. While the expression of Tet07-SIM-mCherry-HoTag6 is induced by dox gradually, the granules will occur abruptly in some time.<br/><br/>
 
  
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                                    <p>The strength of interaction modules can be also controlled. In the rapamycin-induced organelle system, changing the concentration of rapamycin will affect the apparent value of K, a parameter reflecting the interaction strength in our model. In a gradient rapamycin-inducing experiment, the delay time from adding inducer to granules formation was found to be shorter when concentration of rapamycin increases. So we have confirmed the influence of two parameters in models and increased the flexibility of our synthetic organelles.</p>
 
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Figure3 (a) A simulation of organelle formation process in different interaction strength of components.
 
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(b) The speed of FKBP-Frb mediated organelle formation increases with the increasing concentration of rapamycin.
 
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                                    <p>We also tried to characterized other properties, like the liquid-like property of the synthetic organelles, as they may affect the functions. See more details about our characterizations in <a href="https://2018.igem.org/Team:Peking/Phase_Separation_D"/>DataPage Phase separation</a>.</p><br/><br/><br/>
 
 
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                            <div class="texttitle">Functional Organelles
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                                     <p>Since SPOT can form in the cell and be controlled, we go further to consider the functions of SPOT. The functions of SPOT can be descripted in three catalogs: Spatial segmentation, Sensor and metabolic regulation. We verified the spatial segmentation with the condensation of substrates, also we can load the protein we want by fusing it with nanobody. We then verified the sensor with detecting rapamycin and ABA, which shows strong relativity between the concentration and the proportion of yeasts with SPOT. To find the law behind metabolism in the SPOT, we fuse the enzymes that can produce β-carotene into SPOT and measure the difference between with or without SPOT in produce of β-carotene.</p>
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（phase内和phase外的胡萝卜素生产实验）<br/><br/><br/><br/><br/>
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                                     <p>SPOT has been well verified and has various functions. And in the future, this modular system will have great potential in science and practice using. SPOT can change the modules to gain more different properties like diverse inducing method, we can also use it as a platform and then load other protein with some interactions like the interaction between nanobody and GFP. What’s more, we might have the ability to form differernt SPOTs in the cell and regulate them respectively. The functions of SPOT can also diverse. We can build a real time sensor for molecule in living cells to monitoring the concentration changing in environment or in cells. More metabolism pathway can be test in SPOT and we will find some laws of the function of regulate the metabolism. To be summary, more achievement is coming true with SPOT.</p>
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                         <span> &copy;2018 PEKING IGEM. All Rights Reserved.</span>
 
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Latest revision as of 00:27, 18 October 2018

Improvement

Fused with yEGFP

We fused part Frb (BBa_K209496) and FKBP (BBa_K209023) with yEGFP, which made it visible in the yeast under fluorescent microscope. Then we got part Frb-yEGFP (BBa_K2601007) and FKBP-yEGFP (BBa_K2601008) .

Then we characterized the expression of Frb-yEGFP and FKBP-yEGFP with 4 promoters pUra3, pTet07, pTEF1 and PDH3. The expression of these promoters was measured through flow cytometry (Fig. 1).
We made these improved prats:
Tet07-Frb-yEGFP (BBa_K2601021) ;
PDH3-Frb-yEGFP (BBa_K2601023) ;
Tet07-FKBP-yEGFP (BBa_K2601025) ;
TEF1-FKBP-yEGFP (BBa_K2601026) ;
PDH3-FKBP-yEGFP (BBa_K2601027) .



Figure. 1 The strength of three different yeast promoters tested through flow cytometry.




Phase separation system

Moreover, we fused Frb-yEGFP with HOtag6 and FKBP-yEGFP with HOtag3. These parts can form phase separation in the presence of rapamycin (Fig. 2). The original part Frb (BBa_K209496) and FKBP (BBa_K209023) doesn't have this function.
We uploaded 2 parts:
Frb-yEGFP-HOTag6 (BBa_K2601010) ;
FKBP-yEGFP-HOTag3 (BBa_K2601011) .





Figure. 2 The improvement of Frb and FKBP fused with yEGFP.
Figure. 2A The structure of Frb and FKBP. Rapamycin induces the interaction between them.
Figure. 2B Design of RapaSPOT. FKBP is fused with mCherry and HOTag3 while Frb is fused with yEGFP and HOTag6. After adding rapamycin, they are expected self-organizing to form large assemblies, which will be organelles in cells.
Figure. 2C Granules formed in chemical-induced SPOT after adding rapamycin. Ura3-FKBP-HOTag3 with mCherry and PDH3-Frb-HOTag6 with yEGFP are transferred and expressed in S. cerevisiae. Fluorescence images in both GFP and mCherry channels of cells are taken after adding 10 μM Figure. 2D Growth curves of wild-type yeast (black curve) and yeast strains of Ura3-FKBP-HOTag3 and PDH3-Frb-HOTag6 (red curve). They are measured after adding different concentration of rapamycin for 24 hours. Wild-type yeast cannot grow and reproduce normally while recombination yeast strains of FKBP-HOTag3 and Frb-HOTag6 can survive when exposed to rapamycin.


Then we characterized the expression of Frb-yEGFP-HOTag6 with 3 promoters pUra3, pTEF1 and PDH3. As our Modeling work predicts, the kinetics of a system depends on the concentration of the components and the interaction strength(Fig .3). We successfully uploaded 5 parts form them:
pTet07-Frb-yEGFP-HOTag6 (BBa_K2601032) ;
pTEF1-Frb-yEGFP-HOTag6 (BBa_K2601033) ;
PDH3-Frb-yEGFP-HOTag6 (BBa_K2601034) ;
pTEF1-FKBP-yEGFP-HOTag3 (BBa_K2601011) ;
PDH3-yEGFP-HOTag3 (BBa_K2601011) .



Figure. 3 The improvement of Frb and FKBP fused with yEGFP regulated by four kinds of promoters.
Figure. 3A The formation of SPOT can be described as a phase separation process of three components. Two important variables, the concentration of components and the interaction strength are marked in the figure.
Figure. 3B Flow cytometry results of three promoters (pUra3, pTEF2, and PDH3). The expression level of Ura3 is the lowest while PDH3 is the strongest promoter.
Figure. 3C RapaSPOT of different promoter combinations after 10 μM rapamycin induction. Two axes stand for the expression level of components. After 3 hours, only SPOT system with high level of Frb can be observed. Figure. 3D Proportion of yeast with granules after rapamycin induction. The rapamycin concentration range is from 1μM to 100 μM.