Difference between revisions of "Team:Mingdao/InterLab"

 
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       <!-- <img src="https://static.igem.org/mediawiki/2017/1/1b/T--CSMU_NCHU_Taiwan--modeling.png"> -->
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       <img class="top-picture" src="https://static.igem.org/mediawiki/2018/4/45/T--Mingdao--Phil13-8.png" style="width:100%">
 
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             <h1>Structure & Docking Model</h1>
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             <h1>Interlab Study</h1>
 
             <div id="model-intro" class="m-block" >
 
             <div id="model-intro" class="m-block" >
−
                <h2 class="m-subtitle">Introduction</h2>
 
−
                <img src="https://static.igem.org/mediawiki/2017/f/f8/T--CSMU_NCHU_Taiwan--green.png" style="width: 60%; transform: translate(35%, -150%);">
 
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                <h3>What our models do?</h3>
 
−
                <p>CSMU_NCHU Taiwan conducted two modeling projects. The aim of the two dry lab project is to, first, predict the enhanced performance on Aflatoxin degradation of a novel fusion protein, and then try to connect our modeling results back to the lab results, seeking for a reasonable explanation. The main goal is to explain how our protein work and why it has a better performance comparing to the original protein.<br></p>
 
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                <p>The protein we create from the project is a fusion of MSMEG5998 (an aflatoxin degrading protein)<small><small>[1]</small></small> and Thioredoxin (a folding-assisting protein, which can increase the solubility)<small><small>[2]</small></small>. In order to produce accurately folded MSMEG5998, we merge the enzyme with another protein, Thioredoxin, which can improve the performance of protein folding, we expect Thioredoxin can help the fusion protein itself to fold accurately. Therefore, with the fusion protein that we created, our aim is to create a high efficiency protein on degrading aflatoxin.<br></p>
 
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                <p>The modeling project is divided into two parts: Protein structure modeling and docking simulation.<br></p>
 
−
                <p>First, we developed a 3D protein model that can predict the structure of fusion protein and tell us whether the fusion protein is misfolded or not. Since the active sites of MSMEG5998 toward Aflatoxin (ligand) has not been studied, we predict the binding domain of enzyme with Aflatoxin. Then we use the 3D model to simulate the correct binding position, and thus, help us improve the accuracy of fusion protein in wet lab experiments.<br><br></p>
 
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                <p>Our experiment is carried out in two different aspects:</p>
 
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                <p>1.&nbsp;&nbsp;Building the 3D model of the fusion protein</p>
 
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                <p>2.&nbsp;&nbsp;Create a docking simulation of the fusion protein, including the active sites of Thioredoxin and also the binding position of MSMEG5998 with Aflatoxin.<br><br></p>
 
  
−
            </div>
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<h3>Note</h3>
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            <div id="model-protein" class="m-block" >
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<p>Description: the goal and main contents were quoted from iGEM International InterLab Measurement Study <p>
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                <h2 class="m-subtitle">Protein structure modeling</h2>
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Methods: the protocol was provided by iGEM InterLab Committee and described briefly in here <p>
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                <img src="https://static.igem.org/mediawiki/2017/f/f8/T--CSMU_NCHU_Taiwan--green.png" style="width: 60%; transform: translate(35%, -150%);">
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Results: the experiment and data presented here were all made by members of team Mingdao <p>
 +
Reference: <a href="https://2018.igem.org/Measurement/InterLab">Fifth International InterLab Measurement Study@iGEM</a>
 +
 +
</br></br>
 +
<center>
 +
<img src="https://static.igem.org/mediawiki/2018/8/8b/T--Mingdao--Interlablastday1.jpeg" alt="" style="width:49%">
 +
<img src="https://static.igem.org/mediawiki/2018/9/9f/T--Mingdao--Interlablastday2.jpeg" alt="" style="width:49%"></center><br />
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<center><img src="https://static.igem.org/mediawiki/2018/7/75/T--Mingdao--Interlablastday3.jpeg" alt="" style="width:49%">
 +
<img src="https://static.igem.org/mediawiki/2018/e/ef/T--Mingdao--Interlablastday4.jpeg" alt="" style="width:49%">
 +
</center></br>
  
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                <h3>Overview</h3>
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<h3>Instrument</h3>
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<p>The machine in the Biolab of Mingdao High School: Synergy H1 Hybrid Multi-Mode Microplate Reader
 +
<p><img class="center" src="https://static.igem.org/mediawiki/2018/e/e6/T--Mingdao--Interlab0.jpg"alt=""
 +
style="width:80%">
 +
<p>
 +
</br></br>
  
−
                <p>1.&nbsp;&nbsp;The fusion protein is a combination of two different functional proteins: MSMEG5998 and Thioredoxin. The two different proteins are combined by a linker.<small><small>[3]</small></small><br></li>
 
−
                <p>2.&nbsp;&nbsp;The first challenge we’re facing is that there is no existing structure of this protein. The team still manage to predict the model by using similar protein to create a model, the software tool we used is Swiss Model.<small><small>[4][5]</small></small><br></p>
 
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                <p>We only know that MSMEG5998 belongs to FDR-A family, since there is no exact structure of MSMEG5998, so we try to build a reliable model for the purpose below:</p>
 
−
                <p>i.&nbsp;&nbsp;To visualize the stereoscopic structure of the two proteins.</p>
 
−
                <p>ii.&nbsp;To make sure that there is no mutual bonding between the proteins, which can cause misfolding.</p>
 
−
                <br><br>
 
−
                <h3>First of all, we use NCBI to determine the protein sequence we want</h3>
 
−
                <br>
 
−
                <img src="https://static.igem.org/mediawiki/2017/e/e9/T--CSMU_NCHU_Taiwan--model01.png" style="width: 100%">
 
−
                <br><br>
 
−
                <img src="https://static.igem.org/mediawiki/2017/2/27/T--CSMU_NCHU_Taiwan--model02.png" style="width: 100%">
 
−
                <br><br>
 
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                <h3>Next is to insert a linker into the two proteins</h3>
 
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                <p>1.&nbsp;&nbsp;Purpose: Maintain the function of the two protein by separating MSMEG5998 and Thioredoxin.</p>
 
−
                <p>2.&nbsp;&nbsp;The sequence of Linker is: <br>GGTACCCGGGGATCCCTCGAGGGTGGT.</p>
 
  
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                <p>3.&nbsp;&nbsp;The linker our team add has two additional functions:</p>
+
                 <h3>Introduction</h3>
−
                <p>i.&nbsp;&nbsp;Contains four restriction sites: Kpn1, Sma1, BamH1, Xho1.</p>
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                 <p>"Reliable and repeatable measurement is a key component to all engineering disciplines. The same
−
                <p>ii.&nbsp;&nbsp;Two glycine are added at the end of the linker to increase the folding space and the stability, hence lower the chance of misfolding. <small><small>[6]</small></small></p>
+
holds true for synthetic biology, which has also been called engineering biology. However, the
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                <p>iii.&nbsp;&nbsp;Now have a fusion protein with the sequence listed below</p>
+
ability to repeat measurements in different labs has been difficult. The Measurement Committee,
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                <img src="https://static.igem.org/mediawiki/2017/8/81/T--CSMU_NCHU_Taiwan--model03.png" style="width: 100%">
+
through the InterLab study, has been developing a robust measurement procedure for green
−
                <br><br>
+
fluorescent protein (GFP) over the last several years. We chose GFP as the measurement marker
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                <img src="https://static.igem.org/mediawiki/2017/2/21/T--CSMU_NCHU_Taiwan--model04.png" style="width: 100%">
+
for this study since it's one of the most used markers in synthetic biology and, as a result, most
−
                <br><br>
+
laboratories are equipped to measure this protein."  
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                 <h3>Visualize the fusion protein model</h3>
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<p>
−
                 <p>1.&emsp;By using RaptorX[7], the protein sequence can be exported in a PDB file.</p>
+
−
              <br>
+
−
              <img src="https://static.igem.org/mediawiki/2017/c/c4/T--CSMU_NCHU_Taiwan--model05.png" style="width: 100%">
+
−
                <br>
+
−
                <p>2.&emsp;Visualize the structure by using PyMOL.</p>
+
−
              <br>
+
−
              <img src="https://static.igem.org/mediawiki/2017/f/f8/T--CSMU_NCHU_Taiwan--model06.png" style="width: 100%">
+
−
                <br><br>
+
−
                <p>This is the 3D model of the fusion protein, the green structure presented is the backbone of the fusion protein. Notice that it is mainly divided into two area, which are MSMEG5998 and Thioredoxin. The helix is a secondary structure called alpha helix and the flat arrow-like structure is called beta sheet.<br></p>
+
−
                <br><br>
+
−
                <h3>Associate our results with wet lab</h3>
+
−
                <p>After conducting the protein structure modeling, we started to inspect the fusion protein’s function in the wet lab project; that is, to exam whether the fusion protein is performing better than the original MSMEG5998 on degrading Aflatoxin. The assumption toward wet lab project is that since the structure modeling results show no obvious folding error, we speculate the degrading ability toward Aflatoxin is better since Thioredoxin inside the fusion protein might be helping the fusion protein to fold. Please see the wet lab experiments and results here.<br></p>
+
−
            <a href="https://2017.igem.org/Team:CSMU_NCHU_Taiwan/Results#enzyme_function_results" target="_blank">
+
−
              <img class="right" src="https://static.igem.org/mediawiki/2017/6/66/T--CSMU_NCHU_Taiwan--see_more.png" style="width: 15%" alt=""></a>
+
−
            </div>
+
−
            <div id="model-docking" class="m-block" >
+
−
                <h2 class="m-subtitle">Docking modeling</h2>
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−
                <img src="https://static.igem.org/mediawiki/2017/f/f8/T--CSMU_NCHU_Taiwan--green.png" style="width: 60%; transform: translate(22%, -150%);">
+
−
                <h3>Overview</h3>
+
−
                <p>After the team conducted the wet lab experiments on Aflatoxin degradation, the results show a possibility that the two functional parts in the fusion protein may be accurate, therefore, the team want to proof the concept by simulating the binding position of aflatoxin and the fusion protein, in order to assure our fusion protein can be functional or even with a higher performance as expected. The team detected the possible active sites of the proteins in our project and then stimulated the docking process involving the use of AutoDock and PyMol.<small><small>[8]</small></small> By doing so, we are expecting to observe the performance of the fusion protein, and more importantly, to inspect on the improvements from the new protein comparing to the original ones.<font color="#385e66"> Please notice that the fusion protein is merged with two different proteins, which is MEMEG5998 and Thioredoxin. Therefore, in the lateral discussion, docking simulation contains two different protein-ligand model, which are “Thioredoxin-Fusion protein” model and” MSMEG5998-aflatoxinB2” model.</font></p>
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                <h3>The docking simulation of “Thioredoxin-Fusion protein”</h3>
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                <p>1.&nbsp;&nbsp;Since the structure of Thioredoxin has been studied, we can lock down the active site of thioredoxin by use Uniprot. The team found that there are two active site , which are NO. 33 and NO.36 of the sequence.</p>
+
−
                <p>2.&nbsp;&nbsp;By using NCBI BLAST, the team compared the sequence of the fusion protein with Thioredoxin. The team confirmed that the active sites of fusion protein corresponding to the ones of Thioredoxin are No.33 and 36 , both are Cysteine, C.<br><br></p>
+
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                <img src="https://static.igem.org/mediawiki/2017/7/72/T--CSMU_NCHU_Taiwan--m-4-thioredoxin.png" style="width: 100%">
+
−
                <br><br>
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−
                <p>3.&nbsp;&nbsp;The team later constructed a fusion protein 3D model and then labelled the active sites by using PyMOL. By creating the model, the team could learn why thioredoxin is helpful toward protein folding since the active sites of Thioredoxin are not facing away from MSMEG5998.<br><br></p>
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                <img src="https://static.igem.org/mediawiki/2017/7/7e/T--CSMU_NCHU_Taiwan--model08.png" style="width: 100%">
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                <br><br>
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                <p>This 3D model shows the surface of the fusion protein, which allows us to grasp the concept of what our protein looks like. The region labeled in red is the possible binding site of Thioredoxin, which maybe can assist the fusion protein itself or other proteins folding.<br></p>
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                <h3>The structure of the fusion protein (MSMEG5998 part)</h3>
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−
                <p>1.&nbsp;&nbsp;While the structure of MSMEG5998 remains unknown, the team still manage to predict the model by using similar protein to create a model, the software tool we used is Swiss Model<small><small>[3] [4]</small></small>.</p>
+
−
                <p>2.&nbsp;&nbsp;When deciding the model of MEMEG5998, the team used the Swiss Model by comparing the amino acid sequence among the database of protein sequence. There are two main factors lead to two different models, which are by coverage or by identity. The team choose the highest coverage protein sequence to be our model, named” MSMEG5998 Swiss model”.<br><br></p>
+
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            <img src="https://static.igem.org/mediawiki/2017/b/b1/T--CSMU_NCHU_Taiwan--model09.png" style="width: 60%" class="center">
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              <img src="https://static.igem.org/mediawiki/2017/0/02/T--CSMU_NCHU_Taiwan--swiss.png" style="width: 100%">
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                <br><br>
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              <p>3.&nbsp;&nbsp;The sequence of the MSMEG5998 by using Swiss model is compared with that of fusion protein by using Uniprot. The team then discovered three similar groups being labeled below, which are likely active sites.<br><br></p>
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                <img src="https://static.igem.org/mediawiki/2017/0/02/T--CSMU_NCHU_Taiwan--m-8-uniprot.png" style="width: 100%">
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                <br><br>
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                <p>4.&nbsp;&nbsp;The three possible loci corresponding to the fusion protein sequence are:</p>
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                <p>i.&nbsp;&nbsp;189，Arginine，R</p>
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                <p>ii.&nbsp;&nbsp;214，Glutamine，Q</p>
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                <p>iii.&nbsp;246，Alanine，A</p>
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                <p>Since the pdb. files presented by raptorX were unable to visualize hydrogen bonds of the compound, thus the team used PMViewer v1.5.7 to add on hydrogen bonds and negative charge. (the following pictures are compounds before and after enhancements)<br><br></p>
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                <h3>Further enhancements to the compound before docking simulation on MSMEG5998</h3>
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                <img src="https://static.igem.org/mediawiki/2017/7/79/T--CSMU_NCHU_Taiwan--model121.png" style="width: 100%">
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              <p><br>Under PMViewer, the appearance of the protein before enhancements.<br><br></p>
+
  
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              <img src="https://static.igem.org/mediawiki/2017/9/9d/T--CSMU_NCHU_Taiwan--model131.png" style="width: 100%">
 
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              <p><br>The fusion protein after enhancements, which adds hydrogen and charge to the protein. This process allows the structure and the binding process as real as possible.<br></p>
 
  
 +
</ br>
 +
</ br></ br></p>
  
 +
             
 +
         
 +
            <div id="model-goal" class="m-block" >
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                <h3>Goal for the Fifth InterLab</h3>
  
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              <h3>Adding ligand to the docking simulation of MSMEG5998-Aflatoxin B2</h3>
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                <p>"The goal of the iGEM InterLab Study is to identify and correct the sources of systematic variability
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              <p>Search PubChem to locate the ligand, which in this case is AflatoxinB2, and then download the SDF format.<br></p>
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in synthetic biology measurements, so that eventually, measurements that are taken in different
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              <img src="https://static.igem.org/mediawiki/2017/1/1c/T--CSMU_NCHU_Taiwan--m-11-aflatoxin-3.png" style="width: 100%">
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labs will be no more variable than measurements taken within the same lab. Until we reach this
 +
point, synthetic biology will not be able to achieve its full potential as an engineering discipline, as
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labs will not be able to reliably build upon others’ work."
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<p>
  
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              <br><br>
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"This year, teams participating in the interlab study helped iGEM to answer the following
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              <h3>The docking of MSMEG5998 to Aflatoxin B2</h3>
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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?"
 +
<p>
  
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              <p>1.&nbsp;&nbsp;The settings for Aflatoxin B2 before docking:
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Minimize the energy, in order to acquire a stabilized compound which is easier to go through the docking simulation.
+
           
 +
            </div>
 +
                <h3>Calibration Reference</h3>
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 +
                <div id="model-calibration1" class="m-block" >
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                <h2 class="m-subtitle">Calibration 1:OD600 Reference point - LUDOX Protocol</h2>
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<p><span style="background-color: #ccffff;"><strong>Materials</strong></span></p>
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<p>
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<P>1ml LUDOX CL-X
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<p>
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<p>
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ddH2O
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<p>
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<p>
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96 well Black Clear Bottom Plate
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<p>
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<p>
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</P>
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<p>
 +
 
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<p><span style="background-color: #ccffff;"><strong>Method</strong></span></p>
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<p>
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<P>
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&#8595; Add 100 μl LUDOX into wells A1, B1, C1, D1
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<p>
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<p>
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&#8595; Add 100 μl of ddH2 O into wells A2,B2,C2,D2
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<p>
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<p>
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&#8595; Measure absorbance at 600 nm
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<p>
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<p>
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&#8595; Record the data <p>
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<p>
 
</p>
 
</p>
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              <img src="https://static.igem.org/mediawiki/2017/4/4e/T--CSMU_NCHU_Taiwan--model15.png" style="width: 100%">
 
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              <p><br>2.&nbsp;&nbsp;Select the docking function to proceed.</p>
 
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              <h3>Autodocking area</h3>
 
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              <p>The possible autodocking area are limited to the three active sites of MSMEG5998 mentioned earlier, which can increase the model’s accuracy. After autodocking, we visualize the result by using PyMOL to create a 3D docking model. The three active sites for docking are tested, and compared to one another. The team finally come up with one ideal active site, which is 214,glutamine,Q.<br></p>
 
  
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              <img src="https://static.igem.org/mediawiki/2017/1/17/T--CSMU_NCHU_Taiwan--model16.png" style="width: 100%">
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<p>
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              <p><br>The docking was processed by Autodock (please visit our <a href="https://2017.igem.org/Team:CSMU_NCHU_Taiwan/Software" target="_blank">software tools page</a>, the cube area is the area our team choose to process the docking stimulation, the results are in the picture below.<br></p>
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              <img src="https://static.igem.org/mediawiki/2017/c/c9/T--CSMU_NCHU_Taiwan--m-12-214-2.png" style="width: 100%">
+
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              <p><br><br>This is a side view of the protein macromolecule. The MSMEG5998 active site 214 is presented in red, while the blue compound represents Aflatoxin.<br><br></p>
+
  
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            </div>
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<p><span style="background-color: #ccffff;"><strong>Result</strong></span></p>
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            <div id="model-conslusion" class="m-block" >
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<P>
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                <h2 class="m-subtitle">Discussion and Conclusion</h2>
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<p>
−
                <img src="https://static.igem.org/mediawiki/2017/f/f8/T--CSMU_NCHU_Taiwan--green.png" style="width: 60%; transform: translate(35%, -150%);">
+
<img class="center" src="https://static.igem.org/mediawiki/2018/9/9a/T--Mingdao--Modeling--Chart%28img45%29.jpg"alt=""
 +
style="width:80%">
 +
<p>
  
−
                <p>1.&nbsp;&nbsp;By using protein modeling techniques, the team predicted a fusion protein with multifunction while one doesn’t inhibit the other, or creating structural failure. Which later on helped us in the wet lab experiment to proceed.<br></p>
+
<p>The table shows the OD600 measured by a spectrophotometer (see table above) and plate
−
                <p>2.&nbsp;&nbsp;With the software tools, the team is able to predict an enhanced fusion protein (MSMEG5998 combined with Thioredoxin) that performs better than the original protein (MSMEG5998).<br></p>
+
reader data for H2O and LUDOX corresponding to the expected results. The corrected
−
                <p>3.&nbsp;&nbsp;With the cooperation of the wet lab projects, the team is able to confirm the results of the prediction.(Click the button to visit our project’s result.)</p>
+
Abs600 is calculated by subtracting the mean H2O reading. The reference OD600 is defined
−
                <a href="https://2017.igem.org/Team:CSMU_NCHU_Taiwan/Results#antidote" target="_blank">
+
as that measured by the reference spectrophotometer. The correction factor to convert
−
              <img class="right" src="https://static.igem.org/mediawiki/2017/6/66/T--CSMU_NCHU_Taiwan--see_more.png" style="width: 15%" alt=""></a><br>
+
measured Abs600 to OD600 is thus the reference OD600 divided by Abs600. All cell density
−
                <p>4.&nbsp;&nbsp;&nbsp;Future goals:</p>
+
readings using this instrument with the same settings and volume can be converted to
 +
OD600 by multiplying by 4.200.</p>
 +
<p>
  
−
                <p>i.&nbsp;&nbsp;unfortunately, there is a time limit to our project. However, the team would like to continue our modeling project and also put the theory into practice, trying to see whether active site 214 is the actually binding site with Aflatoxin. The team would conduct experiments of point mutation on site 214, to see if the binding affinity changes or not, in order to explain why this site 214 is crucial toward Aflatoxin degradation.</p>
 
−
                <p>ii.&nbsp;&nbsp;After conducting the two main modeling project, our team successfully predicts the function of our fusion protein; however, the long term goal is that the team envisions our aflatoxin-degrading protein put in to massive and commercialized production. <font color="#1c869c"> Therefore, our team would want to measure the productivity of our protein, in order to seek for the ideal producing conditions and reach the maximum efficiency.</font>(Click the button to see some of the results from the experiment our team has conducted.)<br></p>
 
−
            <a href="https://2017.igem.org/Team:CSMU_NCHU_Taiwan/Model/Parts" target="_blank">
 
−
              <img class="right" src="https://static.igem.org/mediawiki/2017/6/66/T--CSMU_NCHU_Taiwan--see_more.png" style="width: 15%" alt=""></a>
 
−
<br>
 
  
 +
<div id="model-calibration2" class="m-block" >
 +
<h2 class="m-subtitle">Calibration 2: Particle Standard Curve - Microsphere Protocol</h2>
 +
<p>
  
−
            </div>
+
<p><span style="background-color: #ccffff;"><strong>Materials</strong></span></p>
−
            <div id="model-references" class="m-block" >
+
<p>
−
                <h2 class="m-subtitle">References</h2>
+
300 μL silica beads Microsphere suspension
−
                <img src="https://static.igem.org/mediawiki/2017/f/f8/T--CSMU_NCHU_Taiwan--green.png" style="width: 60%; transform: translate(35%, -150%);">
+
<p>
 +
<p>
 +
ddH2O
 +
<p>
 +
<p>
 +
96 well Black Clear Bottom Plate
 +
<p>
 +
<p>
 +
</p>
 +
<p>
 +
<p><span style="background-color: #ccffff;"><strong>Method</strong></span></p>
 +
<p>
 +
<p><em><strong>Preparation of the Microsphere stock solution:</strong></em></p>
 +
<p>
 +
<p>
 +
&#8595; Obtain Silica Beads
 +
<p>
 +
&#8595; Pipet 96 μL beads into an eppendorf
 +
<p>
 +
<p>
 +
&#8595; Add 904 μL of ddH2O to the microspheres
 +
<p>
 +
<p>
 +
&#8595; Vortex well to obtain stock Microsphere Solution.
 +
</p>
 +
<p>
 +
&#8595; Preparation of microsphere serial dilutions as follows
 +
<p>
 +
<img class="center" src="https://static.igem.org/mediawiki/2018/b/b0/T--Mingdao--Modeling--SerialDelution%28img47%29.jpg"alt=""
 +
style="width:80%">
 +
<p>
 +
&#8595; Measure Abs 600
 +
<p>
 +
&#8595; Record the data
 +
<p>
 +
<p><span style="background-color: #ccffff;"><strong>Result</strong></span></p>
 +
<p>
 +
<p><em><strong>Raw Data</strong></em></p>
 +
<p>
 +
<p>
 +
<img class="center" src="https://static.igem.org/mediawiki/2018/5/56/T--Mingdao--Modeling--RawData%28img50%29.jpg"alt=""
 +
style="width:80%">
 +
<p>
 +
<p><em><strong>Particle Standard Curve</strong></em></p>
 +
<p>
 +
<p>
 +
<img class="center"src="https://static.igem.org/mediawiki/2018/0/04/T--Mingdao--Interlab4.jpg"alt=""
 +
style="width:80%">
 +
<p>
 +
<p><em><strong>Particle Standard Curve(log scale)</strong></em></p>
 +
<p>
 +
<p>
 +
<img class="center" src="https://static.igem.org/mediawiki/2018/a/ac/T--Mingdao--interlab5.jpg"alt=""
 +
style="width:80%">
 +
<p>
 +
<div id="model-calibration3" class="m-block" >
 +
<h2 class="m-subtitle">Calibration 3: Fluorescence standard curve - Fluorescein Protocol</h2>
 +
<p>
 +
<p><span style="background-color: #ccffff;"><strong>Materials</strong></span></p>
 +
<p>
 +
<p>
 +
Fluorescein (provided in kit)
 +
<p>
 +
<p>
 +
10ml 1xPBS pH 7.4-7.6 (phosphate buffered saline; provided by team)
 +
<p>
 +
<p>
 +
96 well Black Clear Bottom Plate
 +
<p></p>
 +
<p>
 +
<p>
 +
<p><span style="background-color: #ccffff;"><strong>Method</strong></span></p>
 +
<p>
 +
&#8595; Spin down fluorescein kit tube to make sure pellet is at the bottom of tube.
 +
&#8595; Prepare 10x fluorescein stock solution (100 μM) by resuspending fluorescein in 1 mL of 1xPBS.
 +
<p>
 +
&#8595; Dilute the 10x fluorescein stock solution with 1xPBS to make a 1x fluorescein solution with concentration of 10 μM
 +
<p>
 +
&#8595; Prepare the serial dilutions of fluorescein as follows:
 +
<p>
 +
<img class="center" src="https://static.igem.org/mediawiki/2018/0/0b/T--Mingdao--Interlab6.jpg"alt=""
 +
style="width:80%">
 +
<p>
 +
&#8595; Measure fluorescence of all samples in instrument
 +
<p>
 +
&#8595; Record the data
 +
<p>
 +
 
 +
<p><span style="background-color: #ccffff;"><strong>Result</strong></span></p>
 +
<p>
 +
<p><em><strong>Raw Data</strong></em></p>
 +
<p>
 +
<p>
 +
<img class="center" src="https://static.igem.org/mediawiki/2018/3/3c/T--Mingdao--Interlab7.jpg"alt=""
 +
style="width:80%">
 +
<p>
 +
<p><em><strong>Fluorescein Standard Curves</strong></em></p>
 +
<p>
 +
<p>
 +
<img class="center" src="https://static.igem.org/mediawiki/2018/f/f2/T--Mingdao--Interlab8.jpg"alt=""
 +
style="width:80%">
 +
<p>
 +
<p><em><strong>Fluorescein Standard Curves(log scale)</strong></em></p>
 +
<p>
 +
<p>
 +
<img class="center" src="https://static.igem.org/mediawiki/2018/6/69/T--Mingdao--Interlab9.jpg"alt=""
 +
style="width:80%">
 +
<p>
 +
<div id="model-cell" class="m-block" >
 +
<h3>Cell Measurement</h3>
 +
<p>
 +
<p><span style="background-color: #ccffff;"><strong>Materials</strong></span></p>
 +
<p> Competent cells ( Escherichia coli strain DH5 )
 +
<p>
 +
 LB (Luria Bertani) media
 +
<p>
 +
 Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH)
 +
<p>
 +
 50 ml Falcon tube (or equivalent, preferably amber or covered in foil to block light)
 +
<p>
 +
 Incubator at 37°C
 +
<p>
 +
 1.5 ml eppendorf tubes for sample storage
 +
<p>
 +
 Ice bucket with ice
 +
<p>
 +
 Micropipettes and tips
 +
<p>
 +
 96 well Black Clear Bottom Plate
 +
<p></p>
 +
<p>
 +
<p><span style="background-color: #ccffff;"><strong>Workflow</strong></span></p>
 +
<p>
 +
<p>
 +
<img class="center" src="https://static.igem.org/mediawiki/2018/2/22/T--Mingdao--Interlab10.jpg"alt=""
 +
style="width:80%">
 +
<p>
 +
<p><span style="background-color: #ccffff;"><strong>Method</strong></span></p>
 +
<p>
 +
<p>
 +
<img class="center" src="https://static.igem.org/mediawiki/2018/c/c6/T--Mingdao--Interlab11.jpg"alt=""
 +
style="width:80%">
 +
<p>
 +
<p><em><strong>Day1</strong></em></p>
 +
<p>
 +
&#8595; Transform Escherichia coli DH5 with these plasmids
 +
<p>
 +
<p><em><strong>Day2</strong></em></p>
 +
<p>
 +
&#8595; Pick 2 colonies from each group
 +
<p>
 +
&#8595; Inoculate in 5-10 mL LB medium + Cm
 +
<p>
 +
&#8595; Grow the cells overnight (16-18 hours) at 37°C and shake at 220 rpm.
 +
<p>
 +
<p><em><strong>Day 3</strong></em></p>
 +
<p>
 +
&#8595; Make a 1:10 dilution of each overnight culture in LB + Cm by putting 0.5mL of culture into 4.5mL of LB + Cm
 +
<p>
 +
&#8595; Measure Abs 600 of these 1:10 diluted cultures
 +
<p>
 +
&#8595; Record the data
 +
<p>
 +
&#8595; Dilute the cultures further to a target Abs6 00 of 0.02 in a final volume of 12 ml LB medium + Cm in 50 mL tube
 +
<p>
 +
&#8595; Incubate the cultures at 37°C and shake at 220 rpm for 6 hours.
 +
<p>
 +
&#8595; Measure your samples for Abs600 and fluorescence
 +
<p>
 +
&#8595; Record data in your notebook
 +
<p>
 +
<center> Layout for Abs 600 and fluorescence measurement </center>
 +
<p></p>
 +
<p>
 +
<p>
 +
<img class="center" src="https://static.igem.org/mediawiki/2018/1/1a/T--Mingdao--Interlab12.jpg"alt=""
 +
style="width:80%">
 +
<p>
 +
<p><span style="background-color: #ccffff;"><strong>Result</strong></span></p>
 +
<p>
 +
<p><em><strong>Fluorescence Raw Reading</strong></em></p>
 +
<p>
 +
<img class="center" src="https://static.igem.org/mediawiki/2018/2/2b/T--Mingdao--Interlab13.jpg"alt=""
 +
style="width:80%">
 +
<p>
 +
<img class="center" src="https://static.igem.org/mediawiki/2018/6/60/T--Mingdao--Interlab14.jpg"alt=""
 +
style="width:80%">
 +
<p><em><strong>Abs600 Raw Reading</strong></em></p>
 +
<p>
 +
<img class="center" src="https://static.igem.org/mediawiki/2018/2/2d/T--Mingdao--Interlab15.jpg"
 +
<p>
 +
<img class="center" src="https://static.igem.org/mediawiki/2018/4/45/T--Mingdao--Interlab16.jpg">
 +
<div id="model-protocol" class="m-block" >
 +
 
 +
<h3>Colony Forming Units per E. coli cultures at OD600=0.1 </h3>
 +
<p>
 +
&#8595; Measure the OD600 of your cell cultures
 +
<p>
 +
&#8595; Dilute your overnight culture to OD600 = 0.1 in 1mL of LB + Cm media. Do this in triplicate.
 +
<p>
 +
&#8595; Make the following serial dilutions for your triplicates
 +
<p><p>
 +
<img class="center" src="https://static.igem.org/mediawiki/2018/8/8a/T--Mingdao--Interlab19.jpg"alt=""
 +
style="width:80%">
 +
<p>
 +
<p>
 +
&#8595; Aseptically spread plate with 100 μL of the dilutions
 +
<p>
 +
&#8595; Incubate at 37°C overnight
 +
<p>
 +
&#8595; Count colonies after 18-20 hours of growth.
 +
<p>
 +
<p>
  
 +
<p><span style="background-color: #ccffff;"><strong>Result</strong></span></p>
 +
<p>
 +
<p>Colony Forming Units per o.1 OD600 E.coli cultures</p>
 +
<p>
 +
<img class="center" src="https://static.igem.org/mediawiki/2018/0/06/T--Mingdao--Interlab20.jpg"alt=""
 +
style="width:80%">
 +
<img class="center" src="https://static.igem.org/mediawiki/2018/3/36/T--Mingdao--Interlab21.jpg"alt=""
 +
style="width:80%">
  
−
                <div class="pdf-area">
+
         
−
            <span  id="public-btn-1" class="pdfbtn">Click to expand content<i class="fa fa-caret-down" aria-hidden="true"></i></span>
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             <div class="img-container" id="public-1">
 
             <div class="img-container" id="public-1">
−
              <img src="https://static.igem.org/mediawiki/2017/0/0b/T--CSMU_NCHU_Taiwan--modelfinal.png" alt="">
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       <div class="path-tags">
 
       <div class="path-tags">
 
         <ul>
 
         <ul>
−
           <p class="tag">Structure <br>  & Docking Model</p>
+
           <p class="tag">Interlab Study</p>
 
           <li id="intro-btn" class="tag-btn">- Introduction</li>
 
           <li id="intro-btn" class="tag-btn">- Introduction</li>
−
           <li id="protein-btn" class="tag-btn">- Protein Structure Modeling</li>
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           <li id="goal-btn" class="tag-btn">- Goal </li>
−
           <li id="docking-btn" class="tag-btn">- Docking Modeling</li>
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           <li id="calibration1-btn" class="tag-btn">- Calibration 1</li>
−
           <li id="conclusion-btn" class="tag-btn">- Discussion & Conclusion</li>
+
           <li id="calibration2-btn" class="tag-btn">- Calibration 2</li>
 +
          <li id="calibration3-btn" class="tag-btn">- Calibration 3</li>
 +
          <li id="cell-btn" class="tag-btn">- Cell Measurement</li>
 +
          <li id="protocol-btn" class="tag-btn">- Protocol</li>
  
 
           <br>
 
           <br>
−
           <a href="https://2017.igem.org/Team:CSMU_NCHU_Taiwan/Model/Degradation"><p style="font-size:18px;font-family: 'Ubuntu'">Degradation Model</p></a>
+
            
−
          <a href="https://2017.igem.org/Team:CSMU_NCHU_Taiwan/Model/Parts"><p style="font-size:18px;font-family: 'Ubuntu'">Parts Model</p></a>
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     <div class="top">
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       <img src="https://static.igem.org/mediawiki/2018/5/58/T--Mingdao--go_to_top.jpg" alt="">
 
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       $("#goal-btn").click(function() {
 
         $('html, body').animate({
 
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         $('html, body').animate({
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            $("#calibration3-btn").click(function() {
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        $('html, body').animate({
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        }, 500);
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{{:Team:Mingdao/test6}}

Latest revision as of 02:13, 18 October 2018

HOME
Model

Interlab Study

Note

Description: the goal and main contents were quoted from iGEM International InterLab Measurement Study

Methods: the protocol was provided by iGEM InterLab Committee and described briefly in here

Results: the experiment and data presented here were all made by members of team Mingdao

Reference: Fifth International InterLab Measurement Study@iGEM



Instrument

The machine in the Biolab of Mingdao High School: Synergy H1 Hybrid Multi-Mode Microplate Reader



Introduction

"Reliable and repeatable measurement is a key component to all engineering disciplines. The same holds true for synthetic biology, which has also been called engineering biology. However, the ability to repeat measurements in different labs has been difficult. The Measurement Committee, through the InterLab study, has been developing a robust measurement procedure for green fluorescent protein (GFP) over the last several years. We chose GFP as the measurement marker for this study since it's one of the most used markers in synthetic biology and, as a result, most laboratories are equipped to measure this protein."

Goal for the Fifth InterLab

"The goal of the iGEM InterLab Study is to identify and correct the sources of systematic variability in synthetic biology measurements, so that eventually, measurements that are taken in different labs will be no more variable than measurements taken within the same lab. Until we reach this point, synthetic biology will not be able to achieve its full potential as an engineering discipline, as labs will not be able to reliably build upon others’ work."

"This year, teams participating in the interlab study helped iGEM to answer the following 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?"

Calibration Reference

Calibration 1:OD600 Reference point - LUDOX Protocol

Materials

1ml LUDOX CL-X

ddH2O

96 well Black Clear Bottom Plate

Method

↓ Add 100 μl LUDOX into wells A1, B1, C1, D1

↓ Add 100 μl of ddH2 O into wells A2,B2,C2,D2

↓ Measure absorbance at 600 nm

↓ Record the data

Result

The table shows the OD600 measured by a spectrophotometer (see table above) and plate reader data for H2O and LUDOX corresponding to the expected results. The corrected Abs600 is calculated by subtracting the mean H2O reading. The reference OD600 is defined as that measured by the reference spectrophotometer. The correction factor to convert measured Abs600 to OD600 is thus the reference OD600 divided by Abs600. All cell density readings using this instrument with the same settings and volume can be converted to OD600 by multiplying by 4.200.

Calibration 2: Particle Standard Curve - Microsphere Protocol

Materials

300 μL silica beads Microsphere suspension

ddH2O

96 well Black Clear Bottom Plate

Method

Preparation of the Microsphere stock solution:

↓ Obtain Silica Beads

↓ Pipet 96 μL beads into an eppendorf

↓ Add 904 μL of ddH2O to the microspheres

↓ Vortex well to obtain stock Microsphere Solution.

↓ Preparation of microsphere serial dilutions as follows

↓ Measure Abs 600

↓ Record the data

Result

Raw Data

Particle Standard Curve

Particle Standard Curve(log scale)

Calibration 3: Fluorescence standard curve - Fluorescein Protocol

Materials

Fluorescein (provided in kit)

10ml 1xPBS pH 7.4-7.6 (phosphate buffered saline; provided by team)

96 well Black Clear Bottom Plate

Method

↓ Spin down fluorescein kit tube to make sure pellet is at the bottom of tube. ↓ Prepare 10x fluorescein stock solution (100 μM) by resuspending fluorescein in 1 mL of 1xPBS.

↓ Dilute the 10x fluorescein stock solution with 1xPBS to make a 1x fluorescein solution with concentration of 10 μM

↓ Prepare the serial dilutions of fluorescein as follows:

↓ Measure fluorescence of all samples in instrument

↓ Record the data

Result

Raw Data

Fluorescein Standard Curves

Fluorescein Standard Curves(log scale)

Cell Measurement

Materials

 Competent cells ( Escherichia coli strain DH5 )

 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 Black Clear Bottom Plate

Workflow

Method

Day1

↓ Transform Escherichia coli DH5 with these plasmids

Day2

↓ Pick 2 colonies from each group

↓ Inoculate in 5-10 mL LB medium + Cm

↓ Grow the cells overnight (16-18 hours) at 37°C and shake at 220 rpm.

Day 3

↓ Make a 1:10 dilution of each overnight culture in LB + Cm by putting 0.5mL of culture into 4.5mL of LB + Cm

↓ Measure Abs 600 of these 1:10 diluted cultures

↓ Record the data

↓ Dilute the cultures further to a target Abs6 00 of 0.02 in a final volume of 12 ml LB medium + Cm in 50 mL tube

↓ Incubate the cultures at 37°C and shake at 220 rpm for 6 hours.

↓ Measure your samples for Abs600 and fluorescence

↓ Record data in your notebook

Layout for Abs 600 and fluorescence measurement

Result

Fluorescence Raw Reading

Abs600 Raw Reading

Colony Forming Units per E. coli cultures at OD600=0.1

↓ Measure the OD600 of your cell cultures

↓ Dilute your overnight culture to OD600 = 0.1 in 1mL of LB + Cm media. Do this in triplicate.

↓ Make the following serial dilutions for your triplicates

↓ Aseptically spread plate with 100 μL of the dilutions

↓ Incubate at 37°C overnight

↓ Count colonies after 18-20 hours of growth.

Result

Colony Forming Units per o.1 OD600 E.coli cultures

    Interlab Study

  • - Introduction
  • - Goal
  • - Calibration 1
  • - Calibration 2
  • - Calibration 3
  • - Cell Measurement
  • - Protocol