Difference between revisions of "Team:Peking/InterLab"

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                    <div id="logo" style="max-width:0px"><a class="" href="https://2018.igem.org/Team:Peking"><img
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                            <li class="menu-1"><a class="colapse-menu1" href="https://2018.igem.org/Team:Peking">Home</a>
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                                    <li><a href="https://2018.igem.org/Team:Peking/Project" class="barfont1">Description</a></li>
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 +
                            <li class="menu-4"><a class="colapse-menu1" href="https://2018.igem.org/Team:Peking/Software">Software</a>
 +
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                                <li class="dropdown menu-7"><a class="dropdown-toggle" data-toggle="dropdown" href="#" >Achievement</a>
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                                        <li><a href="https://2018.igem.org/Team:Peking/Judging_Form" class="barfont1">Judging Form</a></li>
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 +
                                        <li><a href="https://2018.igem.org/Team:Peking/InterLab" class="barfont1">InterLab</a></li>
  
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                                <li class="dropdown menu-8"><a class="dropdown-toggle" data-toggle="dropdown" href="#" >Lab</a>
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                                        <li><a href="https://2018.igem.org/Team:Peking/Team_Members">Team Members</a></li>
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                                        <li><a href="https://2018.igem.org/Team:Peking/Attributions" class="barfont1">Attributions</a></li>
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+
                                        <li><a href="https://2018.igem.org/Team:Peking/Notebook" class="barfont1">Notebook</a></li>
 +
                                        <li><a href="https://2018.igem.org/Team:Peking/Collaborations" class="barfont1">Collaborations</a></li>
 +
                                        <li><a href="https://2018.igem.org/Team:Peking/Safety" class="barfont1">Safety</a></li>
 +
                                        <li><a href="https://2018.igem.org/Team:Peking/Acknowledgement" class="barfont1">Acknowledgement</a></li>
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 +
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        <!--/Navigation -->
 +
       
 +
       
 +
        <!-- Page Title======================================================================== -->
 +
        <div id="page-title">
 +
            <div class="row">
 +
                <div class="twelve columns centered text-center">
 +
                    <h1>Interlab</h1>
 +
                  <!--  <p class="title1" style="text-align:justify; text-justify:inter-ideograph;">While people are constantly exploring the world, the greatest pursuit is to remould the world. While the ‘phase separation’ in cells is under investigation and in a research boom, the scientific community hopes that the the phenomenon ‘worth of millions of dollars’ can to be artificially designed to enhance original functions and even acquire new functions. Our team, Peking iGEM 2018 go all out to overcome the challenge: artificially designfulfill phase separation in cells and synthesize membraneless organelles.</p> -->
  
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                <section>
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                                    <h4><a href="javascript:void(0);" onclick="naver('A')">&bull;Introduction</a></h4>
 +
                                    <h4><a href="javascript:void(0);" onclick="naver('B')">&bull;Equipment&nbsp;Information</a></h4>
 +
                                    <h4><a href="javascript:void(0);" onclick="naver('C')">&bull;Results</a></h4>
 +
                                    <ul>
 +
                                        <li><a href="javascript:void(0);" onclick="naver('D')">Calibration&nbsp;1</a></li>
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                            <div class="texttitle"><a id="A"></a>Introduction
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                                    <p>Peking 2018 joined the fifth Interlab measurement study. This year we helped to answer this question: Can we reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of OD?
  
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<p> The introduction of this year Interlab:
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                                  Figure. 1 Overall design
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                            <div class="texttitle"><a id="B"></a>Equipment Information
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                                  <!-- <p>To design multivalent modules, it is not ideal to use multiple repeat domains, which not only will make the protein extremely large and bring difficulties to DNA recombination, but also may be problematic for making transgenic animals. Thus, instead of using multiple repeats, we turned to de novo-designed homo-oligomeric coiled coils. And we named these coiled coils as HO-Tag (homo-oligomeric tag). They are short peptides, ~30 amino acids<sup>[1]</sup>, therefore they are ideal tags to introduce multivalency. There are seven coiled coils previously characterized in protein de novo design studies. They have been proved by previous work of Shu Xiaokun’s lab, and according to their work, HOTag3 and HOTag6 are most robust in driving protein droplet formation over a wide range of protein concentrations, so we choose them.
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Plate Reader: Perkin Elemer EnSpire <sup>TM</sup> Multilabel Reader 2300
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Flow Cytometry: BD LSRFortessa <sup>TM</sup> Cell Analyzer
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96 - Well Pate: Corning Incorporated Costar<sup>®️</sup> 3603
  
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<div class="texttitle"><a id="C"></a>Results
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                                    <p>To design interaction modules, we tried a lot of components and we fused them to the N-terminus of HOTag3 or HOTag6. Some of them are spontaneous and some are inducible. And we can regulate them through various kinds of inducers and different intensities of promoters.</p>
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                                    <div class="ordi">1.</div>
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                                    <h3>Calibration 1: OD600 Reference point - LUDOX</h3>
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              style="color: #000000; font-size: x-large"><strong>Peking iGEM 2018</strong></a>
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                                  Figure. 1 The result of LUDOX calibration. The correction factor of our plate reader is 3.316
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                    <a href="https://2018.igem.org/Team:Peking/Design">Design</a>
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                                    <p>Post-translational modifications by the small ubiquitin-like modifier (SUMO) are crucial events in cellular response to radiation and a wide range of DNA-damaging agents. Previous studies have shown that SUMO mediates protein-protein interactions by binding to a SUMO-interacting motif (SIM) on receptor proteins. And recent studies have shown that a protein with ten repeats of human SUMO3 (polySUMO) and a protein with ten repeats of SIM (polySIM) can phase separate in vitro<sup>[3]</sup>. Therefore, we chose SUMO3 and SIM as a pair of interaction modules and they can drive the formation of synthetic organelles spontaneously. For plasmid construction, in order to make synthetic organelles visible, we chose mCherry (red fluorescent protein) and yEGFP (yeast-enhanced green fluorescent protein) as reporters. Then, we fused mCherry between the C-terminus of SIM and the N-terminus of HOTag6. Similarly, we fused yEGFP between the C-terminus of SUMO and the N-terminus of HOTag3. We transformed them into yeast and proved that they can stably express. If it work, we will find red granules colocalize with green granules in cells under fluorescence microscope. </p>
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                                    Figure. 3A Structure of SUMO3 and SIM. Modification of proteins by SUMO are recognized by SUMO-interacting motifs termed SIMs. In natural process, polySUMOylation recruits distinct interaction partners, such as E3 ubiquitin ligases, that bind to polySUMO chains through tandem SIMs. SIMs bind to a surface patch between the α-helix and a β-sheet of the SUMO protein and extend the β-sheet of SUMO by one additional strand. the SIM either attaches as a parallel or an antiparallel strand to the SUMO β-sheet. Binding is primarily mediated by a stretch of four residues containing 3–4 hydrophobic amino acids (I, V, or L). This core interaction motif is a common property of all SIMs<sup>[4]</sup>.
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Figure. 3B Pattern diagram of SUMO-yeGFP and SIM-mCherry. YeGFP is fused to the C-terminus of SUMO and to the N-terminus of HOTag3, mCherry is fused to the C-terminus of SIM and to the N-terminus of HOTag6.
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                                    <div class="ordi">2.</div>
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                                    <h3>Calibration 2: Particle Standard Curve – Microsphere</h3>
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                                  Figure. 2 The result of particle calibration.                             
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                                  Figure. 4 The result of fluorescein calibration.                            
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    <th><img src="https://static.igem.org/mediawiki/2018/e/e8/T--Peking--fluroscencestandcurve2.png"></th>
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    <th><img src="https://static.igem.org/mediawiki/2018/7/7d/T--Peking--fluroscencestandcurve3.png"></th>
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  </tr>
 +
  <tr>
 +
    <th>(a) Fluorescence  Standard Curve - Linear</td>
 +
    <th>(b) Fluorescence Standard Curve - Log Scale</td>
 +
  </tr>
 +
  <tr>
 +
    <th colspan="2"><div align="center">Figure. 5  The result of fluorescence  standard curve
 +
 
 +
</div></td>
 +
  </tr>
 +
</table>
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 +
<p>
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&nbsp;
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</p>
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                                <div class="info">
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                            </div>
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<div class="texttitle"><a id="G"></a>Cell Measurement
  
 
</div>
 
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                            <hr style="border:2px dashed; height:2px" color="#1E90FF">
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                                    <p>Now, we have artificially designed phase separation in cells and synthesized membraneless organelles. But how can we fulfill intended functions with synthetic organelles? Here, we propose two ideas. We reserve two sites to implement functions, which means function modules, such as enzymes in metabolism, proteins in signaling pathway, transcription factors in transcription and so on, have two sites in our designs.</p>
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                <li class="Left"><a href="https://2018.igem.org/Team:Peking/Collaborations"><b>Collaborations</b></a></li><br/><br/>
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                <li class="Left"><a href="https://2018.igem.org/Team:Peking/Acknowledgement">&#9674;&#160;&#160;&#160;Acknowledgement</a><li>
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                <li class="Left"><a href="https://2018.igem.org/Team:Peking/InterLab">>&#160;&#160;&#160;InterLab</a><li>
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    </div>
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<div class="two_thirds_size" >
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<p>
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<p>Welcome to our wiki!</p>
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Materials:
 +
</p>
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<p>
 +
Competent cells (Escherichia coli strain DH5α)
 +
<br/>
 +
LB (Luria Bertani) media
 +
<br/>
 +
Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH)
 +
<br/>
 +
50 ml Falcon tube (or equivalent, preferably amber or covered in foil to block light) Incubator at 37°C
 +
<br/>
 +
1.5 ml eppendorf tubes for sample storage
 +
<br/>
 +
Ice bucket with ice
 +
<br/>
 +
Micropipettes and tips
 +
<br/>
 +
96 well plate, black with clear flat bottom preferred
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</p>
  
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</div>  
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<div align="center"><img src="https://static.igem.org/mediawiki/2018/8/8a/T--Peking--interlabdevice.png" ></div>
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<div id="Content">
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    <figcaption style="text-align:center;">
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<h1>InterLab</h1>
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                                  Figure. 6 The required test devices.                             
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<h2>Calibrations</h2>
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</figcaption> 
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<p>OD600 reference point</p>
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<br/><br/>               
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<p><img src="https://static.igem.org/mediawiki/2018/3/3c/T--Peking--Interlab1.png"/></p>
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</div>         
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<p>Particle Standard Curve</p>
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<br/>
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<p><img src="https://static.igem.org/mediawiki/2018/9/97/T--Peking--Interlab2.png"/></p>
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<div align="center"><img src="https://static.igem.org/mediawiki/2018/5/55/T--Peking--devicelocation.png" ></div>
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<p>Fluorescein Standard Curve</p>
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    <figcaption style="text-align:center;">
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<p><img src="https://static.igem.org/mediawiki/2018/7/71/T--Peking--Interlab3.png"/></p>
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                                  Figure. 7 The localization of each device on the 96-well plate.                             
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<h2>Raw Plate Reader Measurements</h2>
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</figcaption> 
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<p>Fluorescence Raw</p>
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<br/><br/>               
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<p><img src="https://static.igem.org/mediawiki/2018/2/24/T--Peking--Interlab4.png"/></p>
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</div>         
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<p>Abs600 Raw</p>
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<p><img src="https://static.igem.org/mediawiki/2018/c/c0/T--Peking--Interlab5.png"/></p>
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<p>CFU counts</p>
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                            <div class="coll">
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<p><img src="https://static.igem.org/mediawiki/2018/2/2c/T--Peking--Interlab6.png"/></p>
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<a id="H"></a>
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                                    <div class="ordi">1.</div>
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                                </div>
 +
                                <div class="content">
 +
                                    <h3>Plate Reader</h3>
 +
                                </div>
 +
                            </div>
 +
                           
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                            <div class="coll">
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                                <div class="content">
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 +
<div align="center"><img src="https://static.igem.org/mediawiki/2018/5/57/T--Peking--rawplatereadings1.png" ></div>
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<br/>              
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</div>          
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 +
<div align="center"><img src="https://static.igem.org/mediawiki/2018/0/02/T--Peking--rawplatereadings2.png" ></div>
 +
    <figcaption style="text-align:center;">
 +
                                  Figure. 8 The raw readings of Abs 600 and fluorescence by the plate reader.                             
 +
</figcaption> 
 +
 
 +
<br/>
 +
<br/>
 +
 
 +
<div align="center"><img src="https://static.igem.org/mediawiki/2018/e/ec/T--Peking--fluorescence_per_od1.png" ></div>
 +
     
 +
<br/>              
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</div>          
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<div align="center"><img src="https://static.igem.org/mediawiki/2018/6/69/T--Peking--fluorescence_per_od2.png" ></div>
 +
     
 +
<br/>               
 +
</div> 
 +
<div align="center"><img src="https://static.igem.org/mediawiki/2018/7/7b/T--Peking--fluorescence_per_od3.png.png" ></div>
 +
    <figcaption style="text-align:center;">
 +
                                  Figure. 9 The fluorescence per OD.                             
 +
</figcaption>  
 +
<br/>
 +
 
 +
<br/>
 +
 
 +
<div align="center"><img src="https://static.igem.org/mediawiki/2018/1/1a/T--Peking--fluorescence_per_particle1.png" ></div>
 +
     
 +
<br/>              
 +
</div>          
 +
<div align="center"><img src="https://static.igem.org/mediawiki/2018/6/6d/T--Peking--fluorescence_per_particle2.png" ></div>
 +
     
 +
<br/>               
 +
</div> 
 +
<div align="center"><img src="https://static.igem.org/mediawiki/2018/c/c4/T--Peking--fluorescence_per_particle3.png" ></div>
 +
    <figcaption style="text-align:center;">
 +
                                  Figure. 10 The fluorescence per particle.                             
 +
</figcaption>  
 +
<br/>
 +
 
 +
 
 +
                            </div>
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                            <div class="coll">
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                                <div class="info">
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<a id="I"></a>
 +
                                    <div class="ordi">2.</div>
 +
 
 +
                                </div>
 +
                                <div class="content">
 +
                                    <h3>Flow Cytometry</h3>
 +
                                </div>
 +
                            </div>
 +
                           
 +
<br/>
 +
 
 +
<table border="0">
 +
  <tr>
 +
    <th><img src="https://static.igem.org/mediawiki/2018/1/14/T--peking--0hcolony1.png"></th>
 +
    <th><img src="https://static.igem.org/mediawiki/2018/8/81/T--peking--_0hcolony2.png"></th>
 +
  </tr>
 +
  <tr>
 +
    <th>(a) Colony 1</td>
 +
    <th>(b) Colony 2</td>
 +
  </tr>
 +
  <tr>
 +
    <th colspan="2"><div align="center">Figure. 11  The result of flow cytometry at 0h.
 +
 
 +
</div></td>
 +
  </tr>
 +
</table>
 +
 
 +
<br/>
 +
 
 +
<table border="0">
 +
  <tr>
 +
    <th><img src="https://static.igem.org/mediawiki/2018/4/4f/T--peking--_6hcolony1.png"></th>
 +
    <th><img src="https://static.igem.org/mediawiki/2018/d/d7/T--peking--_6hcolony2.png"></th>
 +
  </tr>
 +
  <tr>
 +
    <th>(a) Colony 1</td>
 +
    <th>(b) Colony 2</td>
 +
  </tr>
 +
  <tr>
 +
    <th colspan="2"><div align="center">Figure. 12  The result of flow cytometry at 6h.
 +
 
 +
</div></td>
 +
  </tr>
 +
</table>
 +
 
 +
                            <div class="coll">
 +
 
 +
                                <div class="content">
 +
                                  <!-- <p>We introduce a magic protein, anti-GFP nanobody, which is very small (only 13-kDa, 1.5nm 2.5nm) and high-affinity (0.59nM) camelid antibody to GFP<sup>[8]</sup>. So we can use its characteristic to improve our designs. We can fuse GFP to the C-terminus of interaction modules and to the N-terminus of HOTags, and fuse function modules to the C-terminus of anti-GFP nanobodies. Then, with the help of interaction between anti-GFP nanobodies and GFP, synthetic organelles will “welcome” function modules, expected functions can be realized. You may ask: How does anti-GFP nanobody improve the design? Firstly, it will not make the protein extremely large and will reduce the effect on the structure of function modules, which can ensure the quality of functions. Secondly, it can bring components not belonging to the original structure to synthetic organelles, which can enlarge the enrichment range of synthetic organelles. Thirdly, it is easy to regulate the expression of target proteins. So you can see, nanobodies may do better and give you a surprise!</p>
 +
<div align="center"><img src="https://static.igem.org/mediawiki/2018/e/e5/T--Peking--project_design9.jpeg" width="400px" height="175 px" ></div>
 +
<figcaption style="text-align:center;">
 +
                                  Figure. 7 Interaction of anti-GFP nanobody and GFP
 +
                              </figcaption>
 +
-->
 +
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 +
  <div class="coll">
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 +
                           
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 +
                            <div class="texttitle"><a id="J"></a>Colony Forming Units per 0.1 OD600 E. coli cultures
 
</div>
 
</div>
 +
                            <hr style="border:2px dashed; height:2px" color="#666666">
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<!--
 +
                                    <p>We artificially designed phase separation in cells and synthesized membraneless organelles. And the main work to synthesize an organelle is to fulfill phase separation in a cell, so we stress the importance of interactions and multivalency. For these two aspects, we gave our ideas and the feasibility was analyzed. At last, we proposed two ideas to implement functions. We believe that in the near future, “millions of dollars” will no longer be a dream!</p>
 +
-->
 +
 +
 +
<br/>               
 +
</div> 
 +
<div align="center"><img src="https://static.igem.org/mediawiki/2018/8/8c/T--Peking--CFU.png" ></div>
 +
    <figcaption style="text-align:center;">
 +
                                  Figure. 13 The count of colony forming units per 0.1 OD600 E. coli cultures.                             
 +
</figcaption> 
 +
<br/>
 +
 +
                                </div>
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                            <div class="texttitle"><a id="K"></a>References
 
</div>
 
</div>
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                                    <p>
 +
<a href="https://static.igem.org/mediawiki/2018/0/09/2018_InterLab_Plate_Reader_Protocol.pdf"> [1] https://static.igem.org/mediawiki/2018/0/09/2018_InterLab_Plate_Reader_Protocol.pdf<a/>
 +
</p>
 +
<p>
 +
<a href="https://static.igem.org/mediawiki/2018/e/ec/2018_InterLab_Flow_Cytometry_Protocol.pdf"> [2] https://static.igem.org/mediawiki/2018/e/ec/2018_InterLab_Flow_Cytometry_Protocol.pdf<a/>
 +
<br/>
 +
<br/>
 +
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Latest revision as of 00:28, 18 October 2018

Interlab

Introduction

Peking 2018 joined the fifth Interlab measurement study. This year we helped to answer this question: Can we reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of OD?

The introduction of this year Interlab: https://2018.igem.org/Measurement/InterLab

 

Equipment Information

Plate Reader: Perkin Elemer EnSpire TM Multilabel Reader 2300

Flow Cytometry: BD LSRFortessa TM Cell Analyzer

96 - Well Pate: Corning Incorporated Costar®️ 3603

 

Results

 

1.

Calibration 1: OD600 Reference point - LUDOX

 

Figure. 1 The result of LUDOX calibration. The correction factor of our plate reader is 3.316


 

2.

Calibration 2: Particle Standard Curve – Microsphere

 

Figure. 2 The result of particle calibration.


(a) Particle Standard Curve - Linear (b) Particle Standard Curve - Log Scale
Figure. 3 The result of particle standard curve

 

3.

Calibration 3: Fluorescence standard curve – Fluorescein

 

Figure. 4 The result of fluorescein calibration.


(a) Fluorescence Standard Curve - Linear (b) Fluorescence Standard Curve - Log Scale
Figure. 5 The result of fluorescence standard curve

 

Cell Measurement

 

Materials:

Competent cells (Escherichia coli strain DH5α)
LB (Luria Bertani) media
Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH)
50 ml Falcon tube (or equivalent, preferably amber or covered in foil to block light) Incubator at 37°C
1.5 ml eppendorf tubes for sample storage
Ice bucket with ice
Micropipettes and tips
96 well plate, black with clear flat bottom preferred

Figure. 6 The required test devices.



Figure. 7 The localization of each device on the 96-well plate.


1.

Plate Reader


Figure. 8 The raw readings of Abs 600 and fluorescence by the plate reader.




Figure. 9 The fluorescence per OD.




Figure. 10 The fluorescence per particle.

2.

Flow Cytometry


(a) Colony 1 (b) Colony 2
Figure. 11 The result of flow cytometry at 0h.

(a) Colony 1 (b) Colony 2
Figure. 12 The result of flow cytometry at 6h.
Colony Forming Units per 0.1 OD600 E. coli cultures

Figure. 13 The count of colony forming units per 0.1 OD600 E. coli cultures.

References

[1] https://static.igem.org/mediawiki/2018/0/09/2018_InterLab_Plate_Reader_Protocol.pdf

[2] https://static.igem.org/mediawiki/2018/e/ec/2018_InterLab_Flow_Cytometry_Protocol.pdf