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| − | | + | <!--default for floating navigation--> |
| − | <!--<div class="clear"></div> | + | <div id="comic"> |
| − | | + | <p> |
| − | | + | <!-- <img src="https://static.igem.org/mediawiki/2018/thumb/1/18/T--RDFZ-China--PJDhead1263620.jpg/800px-T--RDFZ-China--PJDhead1263620.jpg" alt="pdcomic" /> --> |
| − | <div class="column full_size">
| + | </p> |
| − | <h1>Improve</h1>
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| − | <p>For teams seeking to improve upon a previous part or project, you should document all of your work on this page. Please remember to include all part measurement and characterization data on the part page on the Registry. Please include a link to your improved part on this page.</p>
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| − | <h3>Gold Medal Criterion #2</h3>
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| − | <p><b>Standard Tracks:</b> Create a new part that has a functional improvement upon an existing BioBrick part. The sequences of the new and existing parts must be different. You must perform experiments with both parts to demonstrate this improvement. Document the experimental characterization on the Part's Main Page on the Registry for both the existing and new parts. Both the new and existing Main Page of each Part’s Registry entry must reference each other. Submit a sample of the new part to the Registry.
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| − | The existing part must NOT be from your 2018 part number range and must be different from the part documented in bronze #4.
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| − | <br><br>
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| − | <b>Special Tracks:</b> Improve the function of an existing iGEM project (that your current team did not originally create) and display your achievement on your wiki.</p>
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| − | <div class="site-logo">
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| − | <img src="https://static.igem.org/mediawiki/2017/5/57/RDFZ_logo.png"> | + | |
| | </div> | | </div> |
| − | | + | <div class="description"> |
| − | <nav class="site-nav">
| + | <h1>Improvement </h1> |
| − |
| + | <div class="topic-title" id="section1"> |
| − | <ul> | + | <p>TlpA is a thermal sensitive transcriptional regulator, original TlpA was provided by ETH Zurich 2017, and a different version with lower activation temperature, TlpA36, was provided by NUS Singapore 2017, is the one we are improving, BBa_K2447012.</p> |
| − | <li class="home">
| + | <p>We provided TlpA39, which is with activation temperature higher than TlpA36 but lower than the original TlpA. All these three proteins can bind to pTlpA promoter, TlpA protein, according to the proposed mechanism, it will dimerize at the operator region of pTlpA, repress it and prevent the RNAP from attach to the promoter. It will de-dimerize when temperature reaches its activation temperature. Leakage was observed, transcription of pTlpA was initiated below the expected temperature. ETH Zurich improved this by simply increase the expression of TlpA protein.</p> |
| − | <a href="https://2018.igem.org/Team:RDFZ-China">HOME</a>
| + | <h4>Why did we improve this?</h4> |
| − |
| + | <p> While we are designing our drug delivery bacteria project, we found out that TlpA has too high activation energy and TlpA36 is too low comparing to human body temperature. So, we found this TlpA39 on Shapiro’s lab, and utilized it into our project. </p> |
| − | </li>
| + | <p> We also constructed a TlpA39-LVA to see if the LVA tag will accelerate the derepression process, but we found that there is no repression to TlpA39 since it has been rapidly degraded.</p> |
| − |
| + | <p> We characterized them by culturing them under different temperature, and read them on a petri dish or plate reader.</ p> |
| − | <li class="project">
| + | <img src="https://static.igem.org/mediawiki/2018 /thumb/8/8e/T--RDFZ-China-- TSRquali. jpeg/ 800px-T--RDFZ-China-- TSRquali.jpeg" width=" 100%"> |
| − | <a href="https://2017.igem.org/Team:RDFZ-China/Description">PROJECT </a>
| + | <p> We can see that at 35 degree Celsius, TlpA36-K2447012 starts to derepress. At 37 degree Celsius, TlpA39- K2572000 starts to derepress. At 39. 5 degree Celsius, Tcl42- K2572001 starts to express. </p> |
| − | <ul>
| + | <p> So they do initiate at different temperature.</ p> |
| − | <li><a href="https://2018.igem.org/Team:RDFZ-China/Description">BACKGROUND</a></li>
| + | <img src="https://static.igem.org/mediawiki/2018/6/ 66/T--RDFZ-China-- TSR.png" width=" 100%"> |
| − | <li><a href="https://2018.igem.org/Team:RDFZ-China/Description">PROJECT</a></li>
| + | <p> Also, we can see there are differences of expression for same regulators are different under different temperature.</p> |
| − | <li><a href="https://2018.igem.org/Team:RDFZ-China/Improve">IMPROVE</a></li>
| + | <p> Piraner, Dan I., et al. "Tunable thermal bioswitches for in vivo control of microbial therapeutics." Nature chemical biology 13.1 ( 2017) : 75.</p> |
| − | </ul>
| + | </div> |
| − | </li> | + | </div> |
| − |
| + | </div> |
| − | <li class="experiment">
| + | |
| − | <a href="https://2017.igem.org/Team:RDFZ-China/Applied_Design">EXPERIMENT</a>
| + | |
| − | <ul>
| + | |
| − | <li><a href="https://2018.igem.org/Team:RDFZ-China/Experiments">EXPERIMENT</a></li>
| + | |
| − | <li><a href="https://2018.igem.org/Team:RDFZ-China/InterLab">INTERLAB</a></li>
| + | |
| − | </ul>
| + | |
| − | </li>
| + | |
| − |
| + | |
| − | <li class="model">
| + | |
| − | <a href="https://2018.igem.org/Team:RDFZ-China/Model">MODEL</a>
| + | |
| − | <ul>
| + | |
| − | <li><a href="https://2018.igem.org/Team:RDFZ-China/Model">MODEL</a></li>
| + | |
| − | <li><a href="https://2018.igem.org/Team:RDFZ-China/Measurement">MEASUREMENT</a></li>
| + | |
| − | </ul>
| + | |
| − | </li>
| + | |
| − |
| + | |
| − | <li class="humanPractice">
| + | |
| − | <a href="https://2018.igem.org/Team:RDFZ-China/Human_Practices">HUMAN<br>PRACTICE</a>
| + | |
| − | <ul>
| + | |
| − | <li><a href="https://2018.igem.org/Team:RDFZ-China/Human_Practices">HUMAN PRACTICE</a></li>
| + | |
| − | <li><a href="https://2018.igem.org/Team:RDFZ-China/Public_Engagement">ENGAGEMENT</a></li>
| + | |
| − | <li><a href="https://2018.igem.org/Team:RDFZ-China/Public_Engagement">GOLD INTEGRATED</a></li>
| + | |
| − | </ul>
| + | |
| − | </li>
| + | |
| − |
| + | |
| − | <li class="demonstrate"><a href=" https://2018.igem.org/Team:RDFZ-China/Demonstrate">DEMONSTRATE</a>
| + | |
| − | <ul>
| + | |
| − | <li><a href=" https://2018.igem.org/Team:RDFZ-China/Demonstrate">DEMONSTRATE</a></li>
| + | |
| − | <li><a href="https://2018.igem.org/Team:RDFZ-China/Applied_Design">APPLIED DESIGN</a></li>
| + | |
| − | </ul>
| + | |
| − | </li>
| + | |
| − |
| + | |
| − | <li class="safety"><a href="https://2018.igem.org/Team:RDFZ-China/Safety">SAFETY</a>
| + | |
| − | </li>
| + | |
| − |
| + | |
| − | <li class="attribution"><a href="https://2018.igem.org/Team:RDFZ-China/Attributions">Attribution</a>
| + | |
| − | <ul>
| + | |
| − | <li><a href="https://2018.igem.org/Team:RDFZ-China/Attributions">ATTRIBUTION</a></li>
| + | |
| − | <li><a href="https://2018.igem.org/Team:RDFZ-China/Collaborations">COLLABORATION</a></li>
| + | |
| − | </ul>
| + | |
| − | </li>
| + | |
| − |
| + | |
| − | <li class="team"><a href="https://2017.igem.org/Team:RDFZ-China/Model">TEAM</a>
| + | |
| − | <ul>
| + | |
| − | <li><a href="https://2018.igem.org/Team:RDFZ-China/Team">MEMBERS</a></li>
| + | |
| − | <li><a href="https://2018.igem.org/Team:RDFZ-China/Team">SCHOOLS</a></li>
| + | |
| − | </ul>
| + | |
| − | </li> | + | |
| − |
| + | |
| − | </ul>
| + | |
| − | </nav>
| + | |
| − | </header>
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| − | | + | |
| − | <div id="topicimg">
| + | |
| − | <img src = "https://static.igem.org/mediawiki/2018/8/8e/T--RDFZ-China--InterLab2.jpg" alt = "Spread plates" width="100%">
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| − | </div>
| + | |
| − | <!--<main>
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| − | <section class = "interlab">
| + | |
| − | <h1 style = "text-align: center">InterLab</h1>
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| − | | + | |
| − | <hr class="line">
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| − | | + | |
| − | <div>
| + | |
| − | <h1>Introduction: What is InterLab</h1>
| + | |
| − | <p> Reliable and repeatable measurement is key to synthetic biology. It is essential for a standard protocol to be established so that the same measurements can be repeated in different labs. This year's interlab study is aimed to reduce the variability of cell count measurements by replacing OD600 measurement which varies between labs with directly counting of colony forming units (CFU) to determine the number of cells in each sample. Then the mean GFP expression level by each cell can be determined by dividing the already standardized GFP expression level of the sample by the number of cells in that sample. | + | |
| − | <br>
| + | |
| − | <br>
| + | |
| − | This is the 5th year of InterLab and we have the following question:
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| − | <br>
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| − | <b>
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| − | <font color = "orange" size = "5" face = "serif">
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| − | 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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| − | </font>
| + | |
| − | </b>
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| − | </p>
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| − |
| + | |
| − | </div>
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| − |
| + | |
| − | <div>
| + | |
| − | <h1>Materials</h1>
| + | |
| − | <p>DNA/Plasmids</p>
| + | |
| − | <ol>
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| − | <li>Negative Control: BBa_R0040</li>
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| − | <li>Positive Control: BBa_I20270</li>
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| − | <li>Test Device 1: BBa_J364000</li>
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| − | <li>Test Device 2: BBa_J364001</li>
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| − | <li>Test Device 3: BBa_J364002</li>
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| − | <li>Test Device 4: BBa_J364007</li>
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| − | <li>Test Device 5: BBa_J364008</li>
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| − | <li>Test Device 6: BBa_J364009</li>
| + | |
| − | </ol>
| + | |
| − | <p>Apparatus</p >
| + | |
| − | <ul>
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| − | <li>96 well plates (provided by Peking University)</li>
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| − | <li>Plate reader</li>
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| − | <li>Foil covered 50 ml tube</li>
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| − | <li>Eppendorf tubes</li>
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| − | <li>Pipettes</li>
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| − | </ul>
| + | |
| − | <p>Materials</p>
| + | |
| − | <ul>
| + | |
| − | <li>LUDOX CL-X</li>
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| − | <li>Silica beads</li>
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| − | <li>Fluorescein</li>
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| − | <li>Phosphate buffered saline</li>
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| − | <li>LB media</li>
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| − | <li>Chloramphenicol</li>
| + | |
| − | <li>LB plates</li>
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| − | <li>distilled water</li>
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| − | </ul>
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| − | </div>
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| − |
| + | |
| − | <div>
| + | |
| − | <h1>Protocols</h1>
| + | |
| − | <div>
| + | |
| − | <p> We followed the protocol provided by iGEM HQ so that inter-laboratory errors can be reduced. Protocols we used can be found here:</p>
| + | |
| − | <a href = "https://static.igem.org/mediawiki/2018/0/09/2018_InterLab_Plate_Reader_Protocol.pdf"> 2018 InterLab Plate Reader Protocol</a><br>
| + | |
| − | <a href = "http://parts.igem.org/Help:Protocols/Transformation">Help: Protocols/Transformation</a>
| + | |
| − | </div>
| + | |
| − | <div>
| + | |
| − | <p>During the first day, we resuspended DNA from distribution kit <em>(Kit Open Day!)</em> and transformed the plasmids into <i>Escherichia coli</i> DH5<i>α</i> competent cells.</p>
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| − | <img src = "https://static.igem.org/mediawiki/2018/6/6b/T--RDFZ-China--InterLab1.jpeg" alt = "Open Distribution Kit" style = "width: 30%">
| + | |
| − | </div>
| + | |
| − | <div>
| + | |
| − | <p> For the second day, firstly we picked single colonies from transformation plates and prepared overnight cultures; we also finished particle and fluorescence calibration.</p> | + | |
| − | <img src = "https://static.igem.org/mediawiki/2018/6/6f/T--RDFZ-China--InterLab5.jpeg" alt = "Add silica beads to 96 well plate" style = "width: 30%">
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| − | </div>
| + | |
| − | <div>
| + | |
| − | <p> The third day was fairly occupied.</p> | + | |
| − | <ol>
| + | |
| − | <li>Overnight cultures were collected for assays. Essentially, each culture was diluted into same starting Abs readings and incubated for 6 hours. Samples were taken at the starting time and after 6 hours. Abs and fluorescence readings were measured for each sample and then imported into excel file.</li>
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| − | <li>While we were waiting for incubation, we diluted the starting sample culture for Colony Forming Units protocols and spread 36 LB plates.</li>
| + | |
| − | <li>We nearly forgot to calibrate OD reference points at the end of the day!</li>
| + | |
| − | </ol>
| + | |
| − | <img src = "https://static.igem.org/mediawiki/2018/8/8e/T--RDFZ-China-- InterLab2. jpg" alt = "Spread plates" width = "30%"> | + | |
| − | <img src = "https://static.igem.org/mediawiki/2018/d/d0/T--RDFZ-China--InterLab3.jpeg" alt = "Add cultures to 96 well plate" width = "30%">
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| − | <img src = "https://static.igem.org/mediawiki/2018/8/83/T--RDFZ-China--InterLab4.jpeg" alt = "With the PLATE READER!" width = "30%">
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| − | </div>
| + | |
| − | <div>
| + | |
| − | <p>At last, we counted the colonies <em>(colony forming units)</em> in those 36 plates. It was just an exhausting process!</p> | + | |
| − | </div>
| + | |
| − | </div>
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| − |
| + | |
| − | <div>
| + | |
| − | <h1>Results</h1>
| + | |
| − | <p><i>The results are shown in the tables and figures.</i></p>
| + | |
| − | <h2>Calibrations</h2>
| + | |
| − | <div class="inlabdiv">
| + | |
| − | <p>OD<sub>600</sub> reference point:</p>
| + | |
| − | <img src = "https://static.igem.org/mediawiki/2018/0/0a/T--RDFZ-China--OD600_Reference_Point.png" alt = "OD600 Reference Point" style = "width: 30%"><br>
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| − | <font size = "1">Table 1. OD600 Reference Point.</font>
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| − | </div>
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| − | | + | |
| − | <div class="inlabdiv">
| + | |
| − | <p>Particle Standard Curve</p>
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| − | <img src = "https://static.igem.org/mediawiki/2018/7/76/T--RDFZ-China--Fluorescein_Standard_Curve.png" alt = "Fluorescein Standard Curve" style = "width: 50%"><br>
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| − | <font size = "1">Figure 1. Particle Standard Curve.</font>
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| − | </div>
| + | |
| − | <div class="inlabdiv">
| + | |
| − | <p>Fluorescein Standard Curve</p>
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| − | <img src = "https://static.igem.org/mediawiki/2018/b/b9/T--RDFZ-China--Particle_Standard_Curve.png" alt = "Particle Standard Curve" style = "width: 50%"><br>
| + | |
| − | <font size = "1">Figure 2. Fluorescein Standard Curve.</font>
| + | |
| − | </div>
| + | |
| − | <h2>Raw Plate Reader Measurements</h2>
| + | |
| − | <div class="inlabdiv">
| + | |
| − | <p> <b>Fluorescence Raw</b></p> | + | |
| − | <img src = "https://static.igem.org/mediawiki/2018/7/74/T--RDFZ-China--Fluorescence_Raw_0_hour.png" alt = "Fluorescence at 0h" style = "width: 50%"><br>
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| − | <font size = "1">Table 2. Raw Plate Reader Measurements of Fluorescence Raw at 0 hour.</font>
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| − | </div>
| + | |
| − | <div class = "inlabdiv">
| + | |
| − | <img src = "https://static.igem.org/mediawiki/2018/ 0/0c/T--RDFZ-China--Fluorescence_Raw_6_hours.png" alt = "Fluorescence at 6h" style = "width: 50%"><br> | + | |
| − | <font size = "1">Table 3. Raw Plate Reader Measurements of Fluorescence Raw at 6 hours.</font>
| + | |
| − | </div>
| + | |
| − | <div class = "inlabdiv">
| + | |
| − | <p><b>Abs<sub>600</sub> Raw</b></p>
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| − | <img src = "https://static.igem.org/mediawiki/2018/c/ce/T--RDFZ-China--Abs600_Raw_0_hour.png" alt = "Abs600 at 0h" style = "width: 50%"><br>
| + | |
| − | <font size = "1">Table 4. Raw Plate Reader Measurements of Abs600 Raw at 0 hour.</font>
| + | |
| − | </div>
| + | |
| − | <div class = "inlabdiv">
| + | |
| − | <img src = "https://static.igem.org/mediawiki/2018/8/89/T--RDFZ-China--Abs600_Raw_6_hours.png" alt = "Abs600 at 6h" style = "width: 50%"><br>
| + | |
| − | <font size = "1">Table 5. Raw Plate Reader Measurements of Abs600 Raw at 6 hours.</font>
| + | |
| − | </div>
| + | |
| − | <div class = "inlabdiv">
| + | |
| − | | + | |
| − | <table style = "width: 70%; height: 200px;">
| + | |
| − | <tr>
| + | |
| − | <th>Device</th>
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| − | <th>Dilution Factor</th>
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| − | <th>CFU Replicate 1</th>
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| − | <th>CFU Replicate 2</th>
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| − | <th>CFU Replicate 3</th>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td rowspan="3">Positive Control 1</td>
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| − | <td>8*10<sup>4</sup></td>
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| − | <td>69</td>
| + | |
| − | <td>22</td>
| + | |
| − | <td>191</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>8*10<sup>5</sup></td>
| + | |
| − | <td>5</td>
| + | |
| − | <td>2</td>
| + | |
| − | <td>5</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>8*10<sup>6</sup></td>
| + | |
| − | <td>1</td>
| + | |
| − | <td>0</td>
| + | |
| − | <td>0</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td rowspan="3">Positive Control 2</td>
| + | |
| − | <td>8*10<sup>4</sup></td>
| + | |
| − | <td>1</td>
| + | |
| − | <td>15</td>
| + | |
| − | <td>65</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>8*10<sup>5</sup></td>
| + | |
| − | <td>1</td>
| + | |
| − | <td>1</td>
| + | |
| − | <td>5</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>8*10<sup>6</sup></td>
| + | |
| − | <td>0</td>
| + | |
| − | <td>0</td>
| + | |
| − | <td>1</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td rowspan="3">Negative Control 1</td>
| + | |
| − | <td>8*10<sup>4</sup></td>
| + | |
| − | <td>98</td>
| + | |
| − | <td>164</td>
| + | |
| − | <td>85</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>8*10<sup>5</sup></td>
| + | |
| − | <td>85</td>
| + | |
| − | <td>29</td>
| + | |
| − | <td>48</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>8*10<sup>6</sup></td>
| + | |
| − | <td>19</td>
| + | |
| − | <td>63</td>
| + | |
| − | <td>23</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td rowspan="3">Negative Control 2</td>
| + | |
| − | <td>8*10<sup>4</sup></td>
| + | |
| − | <td>190</td>
| + | |
| − | <td>226</td>
| + | |
| − | <td>274</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>8*10<sup>5</sup></td>
| + | |
| − | <td>52</td>
| + | |
| − | <td>54</td>
| + | |
| − | <td>49</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>8*10<sup>6</sup></td>
| + | |
| − | <td>78</td>
| + | |
| − | <td>20</td>
| + | |
| − | <td>24</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | <font size = "2">Table 6. Colony Forming Unit Counts.</font>
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| − | </div>
| + | |
| − | </div>
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| − | | + | |
| − | <div>
| + | |
| − | <h1>Evaluation</h1>
| + | |
| − | <p>The control LB measures in Table 4 and Table 5 are a little different, since we changed the LB control after dilution: two different bottle of LB with Chl were measured. The CFU counts in same dilution factor show great variation <b>(Table 6)</b>. High variation may result from the difference of experimenters' spreading methods or fluctuations in the starting sample <em>(starting samples were diluted to OD<sub>600</sub>=0.1 approxiamately)</em>.</p>
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| − | </div>
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| − |
| + | |
| − | <div>
| + | |
| − | <h1>Our Thoughts</h1>
| + | |
| − | <p> <em>Yishen Shen:</em><br>Although it's not my first time to participate in an experiment, I still learned a lot from the InterLab. With so many steps in protocols out there, it can be overwhelming to get started right away. I learned that I should copy down all protocols down on notebook beforehand; it's much easier to follow a protocol that I have written and analyzed by myself. Also, it's handy to obtain all the necessary reagents in advance and make sure they are all in good conditions. I used to run out for reagent in the middle of an experiment and that makes it hectic and messy. Furthermore, I get to know that preparing a timeline is the key. Dividing a day into various blocks keeps me busy and efficient. The three-day experience in InterLab teaches me a lot, though it's mostly repetitive, I got to acquire new knowledge every day and that makes it memorable.</p> | + | |
| − | <p><em>Jianxiang Zhang:</em><br>We feel like that the protocol can sometimes be ambiguous. For instance, the protocol says to "check the OD<sub>600</sub> and make sure it is 0.1 (minus the blank measurement)", it is kind of confusing about how to interpret the content inside the parentheses. Overall, the InterLab study is helpful for providing new measuring methods to our later part characterization study.</p>
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| − | </div>
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| − |
| + | |
| − | <div>
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| − | <h1>Acknowledgements</h1>
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| − | <p><em>Thanks to Molecular Biology Laboratory in Tsinghua University, we could follow the protocols without issues regarding apparatus and reagents. Also, we'd like to appreciate our advisor and anyone who helped us in Tsinghua and Peking University for providing suggestions and guidance during InterLab studies.</em></p>
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