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| − | <script type="text/javascript">
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| + | |
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| | </head> | | </head> |
| | <body> | | <body> |
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| | <div class="fullscreenvideo" > | | <div class="fullscreenvideo" > |
| − | <video id="interlabbg" autoplay="autoplay" muted="muted" loop="loop"> | + | <video id="proofbg" autoplay="autoplay" muted="muted" loop="loop"> |
| | <source src="https://static.igem.org/mediawiki/2018/f/fb/T--NKU_CHINA--BGofother.mp4" type="video/mp4"> | | <source src="https://static.igem.org/mediawiki/2018/f/fb/T--NKU_CHINA--BGofother.mp4" type="video/mp4"> |
| | </video> | | </video> |
| | </div> | | </div> |
| | </div> | | </div> |
| − | <div class="container-fluid">
| + | <main> |
| − | <div class="row">
| + | <h1 class="text-center" style="font-size: 80px;font-weight: normal;color: white;padding-bottom: 0;margin-bottom: 20px; font-family: myTitle;margin-top: 30px;padding-top: 0;">Proof of Concept</h1> |
| − | <div class="component col-xs-4 col-xs-push-1">
| + | |
| − | <div class="subnavpicture" onmouseover="toggleClass()">
| + | <div class="row"> |
| − | <img class="img1" src="https://static.igem.org/mediawiki/2018/3/32/T--NKU_CHINA--cebian1.png" style="height: 250px;width: auto;">
| + | <div class="col-xs-12"> |
| − | <img class="img2" src="https://static.igem.org/mediawiki/2018/7/7e/T--NKU_CHINA--cebian2.png" style="height: 250px;width: auto;">
| + | <p class="proof_content">In order to verify the whole regulation pathway step by step, we divided it into several parts, including the following concepts:</p> |
| − | </div>
| + | <ol class="proof_content"> |
| − | <div class=" round-nav" id="nav-wrapper" onclick="toggledispear()"> | + | <li>Intracellular glutamate can inhibit the <i>gltC</i> expression;</li> |
| − | <ul class="list-unstyled">
| + | <li>GltC can activate the promoter P<sub><i>gltAB</i></sub>;</li> |
| − | <li id="one1"><a href="#firstpart" data-toogle="tooltip" data-placement="right" title="Materials"><span><img src="https://static.igem.org/mediawiki/2018/6/63/T--NKU_CHINA--interlab_subnav1.png" style="width: 50px;height: auto;"></span></a></li>
| + | <li>LacI can inhibit the promoter P<sub><i>grac</i></sub>;</li> |
| − | <li id="two2"><a href="#secondpart" data-toogle="tooltip" data-placement="right" title="Methods"><span><img src="https://static.igem.org/mediawiki/2018/a/a6/T--NKU_CHINA--interlab_subnav2.png" style="width: 50px;height: auto;"></span></a></li>
| + | <li>High glutamate concentration can improve <i>tetA</i> expression.</li> |
| − | <li id="three3"><a href="#thirdpart" data-toogle="tooltip" data-placement="right" title="Results"><span><img src="https://static.igem.org/mediawiki/2018/a/ac/T--NKU_CHINA--interlab_subnav3.png" style="width: 40px;height: auto;"></span></a></li>
| + | </ol> |
| − | </ul>
| + | <p class="proof_content">At last we successively verified the first and the fourth concepts, based on which we achieved the final goal–– control population quality through non-genetic cell-to-cell variation.</p> |
| − | </div> | + | </div> |
| | </div> | | </div> |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − |
| |
| − | <main>
| |
| − | <h3 class="text-center" style="font-size: 80px;font-weight: normal;color: white;padding-bottom: 20px; font-family: myTitle;">Overview</h3>
| |
| − | <p class="homepage-2" style="margin-right: 10%;margin-left: 10%; margin-top:10px; font-size: 20px; color: white;">Poverty in taking reliable and repeatable measurements remains a key obstacle in establishing synthetic biology as an engineering discipline. The Measurement Committee has been studying the measurement procedure for green fluorescent protein (GFP) over the last several years by interlab. The most commonly used markers though GFP is in synthetic biology, labs often resort to making relative comparisons, which makes it difficult for labs to share and data and/or constructs.<br><br>
| |
| − | The goal of the fifth iGEM InterLab Study is to identify and correct the sources of systematic variability in synthetic biology measurements by answering the 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? "<br><br>
| |
| − | As we know in the previous study, the fluorescence value measured by a plate reader is an aggregate measurement of an entire population of cells, we need to divide the total fluorescence by the number of cells in order to determine the mean expression level of GFP per cell. Due to the fact that the "optical density (OD)" of the sample is an approximation of the number of cells varying from lab to lab, we decided to use a special silica beads that are roughly the same size and shape as a typical <i id="firstpart">E. coli</i> cell to set up a universal, standard "equivalent concentration of beads" measurement.
| |
| − | </p>
| |
| − |
| |
| − | <h3 class="text-center" style="font-family: myTitle;font-size: 60px; font-weight: normal;color: white; padding-bottom: 20px;padding-top: 30px;"><img src="https://static.igem.org/mediawiki/2018/6/63/T--NKU_CHINA--interlab_subnav1.png" style="width: 60px;height: auto;">Materials</h3>
| |
| − | <div class="panel-group" id="materials">
| |
| − | <div class="panel panel-default" style="margin-right: 10%;margin-left: 10%;">
| |
| − | <div class="panel-heading panelheadingcursor" data-toggle="collapse" data-parent="#materials" data-target="#collapse1" style="background-color: #B5CACF;">
| |
| − | <h4 class="panel-title" style="height: 35px;">
| |
| − | <a style="font-size: 25px;">Reagents and Apparatus</a>
| |
| − | </h4>
| |
| − | </div>
| |
| − | <div id="collapse1" class="panel-collapse collapse">
| |
| − | <div class="panel-body">
| |
| − | <ul class="interlab-list">
| |
| − | <li style="font-size: 20px;line-height: 25px;">Competent cells (<i>Escherichia coli</i> strain DH5α)</li>
| |
| − | <li style="font-size: 20px;line-height: 25px;">1 mL LUDOX CL-X (provided in kit) </li>
| |
| − | <li style="font-size: 20px;line-height: 25px;">300 µL Silica beads - Microsphere suspension (provided in kit, 4.7 x 10<sup>8</sup> microspheres)</li>
| |
| − | <li style="font-size: 20px;line-height: 25px;">LB (Luria Bertani) media</li>
| |
| − | <li style="font-size: 20px;line-height: 25px;">Fluorescein (provided in kit) </li>
| |
| − | <li style="font-size: 20px;line-height: 25px;">10 mL 1xPBS pH 7.4-7.6 (phosphate buffered saline; provided by team) </li>
| |
| − | <li style="font-size: 20px;line-height: 25px;">Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH) </li>
| |
| − | <li style="font-size: 20px;line-height: 25px;">50 mL Falcon tube (or equivalent, preferably amber or covered in foil to block light) </li>
| |
| − | <li style="font-size: 20px;line-height: 25px;">Incubator at 37°C</li>
| |
| − | <li style="font-size: 20px;line-height: 25px;">1.5 mL eppendorf tubes for sample storage</li>
| |
| − | <li style="font-size: 20px;line-height: 25px;">Ice bucket with ice</li>
| |
| − | <li style="font-size: 20px;line-height: 25px;">Micropipettes and tips</li>
| |
| − | <li style="font-size: 20px;line-height: 25px;">96 well plate, black with clear flat bottom preferred (provided by team)</li>
| |
| − | </ul>
| |
| | </div> | | </div> |
| − | </div>
| + | |
| − | </div>
| + | |
| − | | + | <div class="col-xs-12"> |
| − | <div class="panel panel-default" style="margin-right: 10%;margin-left: 10%;"> | + | <img src="https://static.igem.org/mediawiki/2018/3/34/T--NKU_CHINA--proof1.png" class="img-responsive center-block" style="height: 50px;width: auto;"> |
| − | <div class=" panel-heading panelheadingcursor" data-toggle="collapse" data-parent="#materials" data-target="#collapse2" style="background-color: #D5E1E4;"> | + | </div> |
| − | <h4 class="panel-title" style="height: 35px;" id="secondpart">
| + | </div> |
| − | <a style="font-size: 25px;">Devices</a>
| + | |
| − | </h4>
| + | |
| − | </div>
| + | |
| − | <div id="collapse2" class="panel-collapse collapse">
| + | |
| − | <div class="panel-body">
| + | |
| − | <h5 style="font-size: 20px;line-height: 25px;">From Distribution Kit, all in pSB1C3 backbone:</h5>
| + | |
| − | <ul class="interlab-list">
| + | |
| − | <li style="font-size: 20px;line-height: 25px;">Negative control BBa_R0040</li>
| + | |
| − | <li style="font-size: 20px;line-height: 25px;">Positive control BBa_I20270</li>
| + | |
| − | <li style="font-size: 20px;line-height: 25px;">Test Device 1 BBa_J364000</li>
| + | |
| − | <li style="font-size: 20px;line-height: 25px;">Test Device 2 BBa_J364001</li>
| + | |
| − | <li style="font-size: 20px;line-height: 25px;">Test Device 3 BBa_J364002</li>
| + | |
| − | <li style="font-size: 20px;line-height: 25px;">Test Device 4 BBa_J364007</li>
| + | |
| − | <li style="font-size: 20px;line-height: 25px;">Test Device 5 BBa_J364008</li>
| + | |
| − | <li style="font-size: 20px;line-height: 25px;">Test Device 6 BBa_J364009</li>
| + | |
| − | </ul>
| + | |
| | </div> | | </div> |
| − | </div>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | </div>
| + | <p class="proof_content">According to previous research, <i>B. amyloliquefaciens</i> LL3 is a glutamic acid- independent poly-γ-glutamate (γ-PGA)-producing strain which was isolated from traditional fermented food. Its <i>gltAB</i> gene encodes the glutamate synthase. The increasing intracellular glutamate concentration has a negative effect on the expression of <i>gltAB</i> via modulating GltC activity.</p> |
| − | | + | <p class="proof_content">As a result, the transcription of <i>gltC</i> should be inhibited while intercellular glutamate concentration increasing. We verified this concept by transformed our PopQC system pHT01-mCherry-LacI-P<sub><i>gltAB</i></sub>-P<sub><i>grac</i></sub>-TetA-GFP<sup>int</sup> into <i>B. amyloliquefaciens</i> LL3. Then we measured its intracellular glutamate concentration and transcriptional level of <i>gltC</i> under different concentration of glutamate in fermentation medium. The results proved our assumption to be correct.</p> |
| − | <h3 class="text-center" style="font-family: myTitle;font-size: 60px;font-weight: normal;color: white; padding-bottom: 20px;padding-top: 30px;"><img src="https://static.igem.org/mediawiki/2018/a/a6/T--NKU_CHINA--interlab_subnav2.png" style="width: 60px;height: auto;">Methods</h3>
| + | </div> |
| − | <div class="panel-group" id="methods"> | + | </div> |
| − | <div class=" panel panel- default" style="margin- right: 10%;margin-left: 10%;"> | + | |
| − | <div class="panel-heading panelheadingcursor" data-toggle="collapse" data-parent="#methods" data-target="#collapse3" style="background-color: #89B777;">
| + | |
| − | <h4 class="panel-title" style="height: 35px;">
| + | |
| − | <a style="font-size: 25px;">OD<sub>600</sub> Reference Point</a>
| + | |
| − | </h4>
| + | |
| − | </div>
| + | |
| − | <div id="collapse3" class="panel-collapse collapse">
| + | |
| − | <div class="panel-body">
| + | |
| − | <ul class="interlab-list">
| + | |
| − | <li style="font-size: 20px;line-height: 25px;">Add 100 µL LUDOX into wells A1, B1, C1, D1</li>
| + | |
| − | <li style="font-size: 20px;line-height: 25px;">Add 100 µL of dd H<sub>2</sub>O into wells A2, B2, C2, D2</li>
| + | |
| − | <li style="font-size: 20px;line-height: 25px;">Measure absorbance at 600 nm of all samples in the measurement mode you plan to use for cell measurements</li>
| + | |
| − | <li style="font-size: 20px;line-height: 25px;">Record the data in the table below or in your notebook</li>
| + | |
| − | <li style="font-size: 20px;line-height: 25px;">Import data into Excel sheet provided (OD<sub>600</sub> reference point tab)</li>
| + | |
| − | </ul>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | <div class="panel panel-default" style="margin-right: 10%;margin-left: 10%;">
| + | |
| − | <div class="panel-heading panelheadingcursor" data-toggle="collapse" data-parent="#methods" data-target="#collapse4" style="background-color: #95BF85;">
| + | |
| − | <h4 class="panel-title" style="height: 35px;">
| + | |
| − | <a style="font-size: 25px;">Particle Standard Curve</a>
| + | |
| − | </h4>
| + | |
| − | </div>
| + | |
| − | <div id="collapse4" class="panel-collapse collapse">
| + | |
| − | <div class="panel-body">
| + | |
| − | <ul class="interlab-list">
| + | |
| − | <li style="font-size: 20px;line-height: 25px;">Obtain the tube labeled "Silica Beads" from the InterLab test kit and vortex vigorously for 30 seconds</li>
| + | |
| − | <li style="font-size: 20px;line-height: 25px;">Immediately pipet 96 µL microspheres into a 1.5 mL eppendorf tube</li>
| + | |
| − | <li style="font-size: 20px;line-height: 25px;">Add 904 µL of ddH<sub>2</sub>O to the microspheres</li>
| + | |
| − | <li style="font-size: 20px;line-height: 25px;">Vortex well. This is your Microsphere Stock Solution</li>
| + | |
| − | <li style="font-size: 20px;line-height: 25px;">Repeat dilution series for rows B, C, D</li>
| + | |
| − | <li style="font-size: 20px;line-height: 25px;">Re-Mix (Pipette up and down) each row of plate immediately before putting in the plate reader</li>
| + | |
| − | <li style="font-size: 20px;line-height: 25px;">Measure Abs<sub>600</sub> of all samples in instrument</li>
| + | |
| − | <li style="font-size: 20px;line-height: 25px;">Record the data in your notebook</li>
| + | |
| − | <li style="font-size: 20px;line-height: 25px;">Import data into Excel sheet provided (particle standard curve tab)</li>
| + | |
| − | </ul> | + | |
| | </div> | | </div> |
| − | </div>
| + | |
| − | </div>
| + | |
| − | | + | <div class="col-xs-12"> |
| − | <div class=" panel panel-default" style="margin-right: 10%;margin-left: 10%;"> | + | <img src="https://static.igem.org/mediawiki/2018/f/fa/T--NKU_CHINA--gltC.png" class="img-responsive center-block" style="border-radius: 5px;"> |
| − | <div class=" panel-heading panelheadingcursor" data-toggle="collapse" data-parent="#methods" data-target="#collapse5" style="background-color: #ADCEA1;"> | + | <p class="tuzhu"><strong>Fig. 1. The intracellular glutamate concentration and the relative expression level of <i>gltC</i> in LL3 with P<sub><i>gltAB</i></sub>-GFP in plateau stage. a. The intracellular glutamate concentration of LL3 with P<sub><i>gltAB</i></sub>-GFP in plateau stage. *Significantly different (P < 0.05) by Student's t-test. b. The relative expression level of <i>gltC</i> in plateau stage.</strong> The value illustrates the effect of glutamate concentration on the transcription of <i>gltC</i>. ***Very very significantly different (P < 0.005) by Student's t-test. The strains were cultured at 37 °C in M9 medium with 5 µg/mL chloromycetin under different extracellular glutamate concentration (0 g/L, 2.5 g/L, 5.0 g/L) for 24 hours. Data indicate mean values ± standard deviations from three independent experiments performed in triplicates.</p> |
| − | <h4 class="panel-title" style="height: 35px;">
| + | </div> |
| − | <a style="font-size: 25px;">Fluorescence Standard Curve</a>
| + | </div> |
| − | </h4>
| + | |
| − | </div>
| + | |
| − | <div id="collapse5" class="panel-collapse collapse">
| + | |
| − | <div class="panel-body">
| + | |
| − | <ul class="interlab-list">
| + | |
| − | <li style="font-size: 20px;line-height: 25px;">Spin down fluorescein kit tube to make sure pellet is at the bottom of tube. </li>
| + | |
| − | <li style="font-size: 20px;line-height: 25px;">Prepare 10x fluorescein stock solution (100 µM) by resuspending fluorescein in 1 mL of 1xPBS</li>
| + | |
| − | <li style="font-size: 20px;line-height: 25px;">Dilute the 10x fluorescein stock solution with 1xPBS to make a 1x fluorescein solution with concentration 10 µM: 100 µL of 10x fluorescein stock into 900 µL 1xPBS</li> | + | |
| − | <li style="font-size: 20px;line-height: 25px;">Add 100 µL of PBS into wells A2, B2, C2, D2...A12, B12, C12, D12</li> | + | |
| − | <li style="font-size: 20px;line-height: 25px;">Add 200 µL of fluorescein 1x stock solution into A1, B1, C1, D1</li> | + | |
| − | <li style="font-size: 20px;line-height: 25px;">Transfer 100 µL of fluorescein stock solution from A1 into A2</li>
| + | |
| − | <li style="font-size: 20px;line-height: 25px;">Mix A2 by pipetting up and down 3x and transfer 100 µL into A3...</li>
| + | |
| − | <li style="font-size: 20px;line-height: 25px;">Mix A3 by pipetting up and down 3x and transfer 100 µL into A4...</li>
| + | |
| − | <li style="font-size: 20px;line-height: 25px;">Mix A4 by pipetting up and down 3x and transfer 100 µL into A5...</li> | + | |
| − | <li style="font-size: 20px;line-height: 25px;">Mix A5 by pipetting up and down 3x and transfer 100 µL into A6...</li> | + | |
| − | <li style="font-size: 20px;line-height: 25px;">Mix A6 by pipetting up and down 3x and transfer 100 µL into A7...</li>
| + | |
| − | <li style="font-size: 20px;line-height: 25px;" style="font-size: 20px;line-height: 25px;">Mix A7 by pipetting up and down 3x and transfer 100 µL into A8...</li>
| + | |
| − | <li style="font-size: 20px;line-height: 25px;">Mix A8 by pipetting up and down 3x and transfer 100 µL into A9...</li>
| + | |
| − | <li style="font-size: 20px;line-height: 25px;">Mix A9 by pipetting up and down 3x and transfer 100 µL into A10...</li>
| + | |
| − | <li style="font-size: 20px;line-height: 25px;">Mix A10 by pipetting up and down 3x and transfer 100 µL into A11...</li>
| + | |
| − | <li style="font-size: 20px;line-height: 25px;">Mix A11 by pipetting up and down 3x and transfer 100 µL into liquid waste</li>
| + | |
| − | <li style="font-size: 20px;line-height: 25px;">Repeat dilution series for rows B, C, D</li>
| + | |
| − | <li style="font-size: 20px;line-height: 25px;">Measure fluorescence of all samples in instrument</li>
| + | |
| − | <li style="font-size: 20px;line-height: 25px;">Record the data in your notebook</li>
| + | |
| − | <li style="font-size: 20px;line-height: 25px;">Import data into Excel sheet provided (fluorescein standard curve tab)</li>
| + | |
| − | </ul>
| + | |
| | </div> | | </div> |
| − | </div>
| + | |
| − | </div>
| + | <div class="row"> |
| − | | + | |
| − | <div class="panel panel-default" style="margin-right: 10%;margin-left: 10%;"> | + | <img src="https://static.igem.org/mediawiki/2018/4/4d/T--NKU_CHINA--proof2.png" class="img-responsive center-block" style="height: 50px;width: auto;"> |
| − | <div class="panel-heading panelheadingcursor" data-toggle="collapse" data-parent="#methods" data-target="#collapse6" style="background-color: #C3DBBA;">
| + | </div> |
| − | <h4 class="panel-title" style="height: 35px;">
| + | </div> |
| − | <a style="font-size: 25px;">Competent Cells and Transformation</a> | + | |
| − | </h4>
| + | |
| − | </div>
| + | |
| − | <div id="collapse6" class="panel-collapse collapse">
| + | |
| − | | + | |
| − | <ul class="interlab-list">
| + | |
| − | <li style="font-size: 20px;line-height: 25px;">Resuspend DNA in selected wells in the Distribution Kit with 10 µL dH<sub>2</sub>O</li>
| + | |
| − | <li style="font-size: 20px;line-height: 25px;">Thaw competent cells on ice</li>
| + | |
| − | <li style="font-size: 20px;line-height: 25px;">Pipette 50 µL of competent cells into 1.5 mL tube</li>
| + | |
| − | <li style="font-size: 20px;line-height: 25px;">Pipette 1 µL of resuspended DNA into 1.5 mL tube</li>
| + | |
| − | <li style="font-size: 20px;line-height: 25px;">Pipette 1 µL of control DNA into 2 mL tube</li>
| + | |
| − | <li style="font-size: 20px;line-height: 25px;">Close 1.5 mL tubes, incubate on ice for 30 min</li>
| + | |
| − | <li style="font-size: 20px;line-height: 25px;">Heat shock tubes at 42°C for 45 sec</li>
| + | |
| − | <li style="font-size: 20px;line-height: 25px;">Incubate on ice for 5 min</li>
| + | |
| − | <li style="font-size: 20px;line-height: 25px;">Pipette 950 µL SOC media to each transformation</li>
| + | |
| − | <li style="font-size: 20px;line-height: 25px;">Incubate at 37°C for 1 hours, shaking at 200-300 rpm</li>
| + | |
| − | <li style="font-size: 20px;line-height: 25px;">Pipette 100 µL of each transformation onto petri plates</li>
| + | |
| − | <li style="font-size: 20px;line-height: 25px;">Spin down cells at 6800 g for 3 min and discard 800 µL of the supernatant. Resuspend the cells in the remaining 100 µL, and pipette each transformation onto petri plates</li>
| + | |
| − | <li style="font-size: 20px;line-height: 25px;">Incubate transformations overnight (14-18 hr) at 37°C</li>
| + | |
| − | <li style="font-size: 20px;line-height: 25px;">Pick single colonies for PCR</li>
| + | |
| − | <li style="font-size: 20px;line-height: 25px;">Count colonies for control transformation</li>
| + | |
| − | </ul>
| + | |
| | </div> | | </div> |
| − | </div>
| + | |
| − | </div>
| + | <div class="row"> |
| − | | + | |
| − | <div class="panel panel-default" style="margin-right: 10%;margin-left: 10%;"> | + | <p class="proof_content">On the basis of the above experiment, we intended to choose GFP as a signal for P<sub><i>gltAB</i></sub> activity, so that we can easily measure the florescence intensity by plate reader, combining with the transcriptional level of <i>gltC</i> measured at the same time. Due to time constraints, we didn't proof this concept.</p> |
| − | <div class="panel-heading panelheadingcursor" data-toggle="collapse" data-parent="#methods" data-target="#collapse7" style="background-color: #D3E5CE;">
| + | </div> |
| − | <h4 class="panel-title" style="height: 35px;">
| + | </div> |
| − | <a style="font-size: 25px;">Cell Measurement</a> | + | |
| − | </h4>
| + | |
| − | </div>
| + | |
| − | <div id="collapse7" class="panel-collapse collapse">
| + | |
| − | | + | |
| − | <ul class="interlab-list">
| + | |
| − | <li style="font-size: 20px;line-height: 25px;">Pick 2 colonies from each of the transformation plates and inoculate in 5-10 mL LB medium + Chloramphenicol. Grow the cells overnight (16-18 hours) at 37°C and 220 rpm</li>
| + | |
| − | <li style="font-size: 20px;line-height: 25px;">Make a 1:10 dilution of each overnight culture in LB + Chloramphenicol (0.5 mL of culture into 4.5 mL of LB + Chlor)</li>
| + | |
| − | <li style="font-size: 20px;line-height: 25px;">Measure Abs<sub>600</sub> of these 1:10 diluted cultures</li>
| + | |
| − | <li style="font-size: 20px;line-height: 25px;">Record the data in your notebook</li>
| + | |
| − | <li style="font-size: 20px;line-height: 25px;">Dilute the cultures further to a target Abs<sub>600</sub> of 0.02 in a final volume of 12 mL LB medium + Chloramphenicol in 50 mL falcon tube (amber, or covered with foil to block light)</li>
| + | |
| − | <li style="font-size: 20px;line-height: 25px;">Take 500 µL samples of the diluted cultures at 0 hours into 1.5 mL Eppendorf tubes, prior to incubation. (At each time point 0 hours and 6 hours, you will take a sample from each of the 8 devices, two colonies per device, for a total of 16 Eppendorf tubes with 500 µL samples per time point, 32 samples total). Place the samples on ice</li> | + | |
| − | <li style="font-size: 20px;line-height: 25px;">Incubate the remainder of the cultures at 37°C and 220 rpm for 6 hours</li>
| + | |
| − | <li style="font-size: 20px;line-height: 25px;">Take 500 µL samples of the cultures at 6 hours of incubation into 1.5 mL Eppendorf tubes. Place samples on ice</li>
| + | |
| − | <li style="font-size: 20px;line-height: 25px;">At the end of sampling point you need to measure your samples (Abs<sub>600</sub> and fluorescence measurement), see the below for details</li>
| + | |
| − | <li style="font-size: 20px;line-height: 25px;">Record data in your notebook</li>
| + | |
| − | <li style="font-size: 20px;line-height: 25px;">Import data into Excel sheet provided (fluorescence measurement tab)</li>
| + | |
| − | </ul>
| + | |
| | </div> | | </div> |
| − | </div>
| + | |
| − | </div>
| + | |
| − | | + | <div class="col-xs-12"> |
| − | <div class=" panel panel-default" style="margin-right: 10%;margin-left: 10%;"> | + | <img src="https://static.igem.org/mediawiki/2018/5/5b/T--NKU_CHINA--proof3.png" class="img-responsive center-block" style="height: 50px;width: auto;"> |
| − | <div class=" panel-heading panelheadingcursor" data-toggle="collapse" data-parent="#methods" data-target="#collapse8" style="background-color: #E7F1E4;"> | + | </div> |
| − | <h4 class="panel-title" style="height: 35px;" id="thirdpart">
| + | </div> |
| − | <a style="font-size: 25px;">CFU per 0.1 OD<sub>600</sub> <i>E.coli</i> Cultures</a>
| + | |
| − | </h4>
| + | |
| − | </div>
| + | |
| − | <div id="collapse8" class="panel-collapse collapse">
| + | |
| − | <div class="panel-body">
| + | |
| − | <ul class="interlab-list">
| + | |
| − | <li style="font-size: 20px;line-height: 25px;">Culture colonies for two Positive Control (BBa_I20270) cultures and your two Negative Control (BBa_R0040) cultures for 16-18 hours</li>
| + | |
| − | <li style="font-size: 20px;line-height: 25px;">Dilute the overnight culture to OD<sub>600</sub> = 0.1 in 1 mL of LB + Cam media. Do this in triplicate for each culture. Check the OD<sub>600</sub> and make sure it is 0.1</li>
| + | |
| − | <li style="font-size: 20px;line-height: 25px;">Aseptically spead plate 100 µL on LB + Cam plates for those Final Dilution Factor is 8 x 10<sup>4</sup> or 8 x 10<sup>5</sup> or 8 x 10<sup>6</sup></li>
| + | |
| − | <li style="font-size: 20px;line-height: 25px;">Incubate at 37°C overnight and count colonies after 18-20 hours of growth</li>
| + | |
| − | <li style="font-size: 20px;line-height: 25px;">Count the colonies on each plate with fewer than 300 colonies. Multiple the colony count by the Final Dilution Factor on each plate</li>
| + | |
| − | </ul> | + | |
| | </div> | | </div> |
| − | </div>
| + | |
| − | </div>
| + | <div class="row"> |
| − | </div>
| + | |
| − | </div>
| + | <p class=" proof_content"> As we all know, LacI plays an important role in lac operon model, it can sufficiently inhibit the activity of its downstream promoters. For < i> Bacillus</ i>, the promoter P< sub>< i> grac</ i></sub> consists of the < i> groE</ i> promoter; the < i> lacO</ i> operator and the < i> gsiB</ i> SD sequence, among which the
the < i> lacO</ i> operator can be inhibited by the LacI protein. Due to time constraints, we didn&# 39; t proof this concept.</p> |
| − | | + | <p class="center_content">High glutamate concentration can improve <i>tetA</i> expression</p> |
| − | <h3 class="text-center" style="font-family: myTitle;font-size: 60px;font-weight: normal;color: white;padding-bottom: 20px;padding-top: 30px;"><img src="https://static.igem.org/mediawiki/2018/a/ac/T--NKU_CHINA--interlab_subnav3.png" style="width: 60px;height: auto;">Results</h3>
| + | <p class="proof_content">To test the expression of <i>tetA</i>, we tagged it with the fluorescent reporter GFP-coding gene (<a href="http://parts.igem.org/Part:BBa_K2705004" style="color: orange;">BBa_K2705004</a>), whose expression was detected by microplate assay (395 nm\509 nm). The intracellular glutamate concentration and bacteria concentration (OD<sub>600</sub>) were also examined, respectively. (See <strong>Figure 2.</strong>) It could be concluded that with the increasing glutamate in medium, intracellular glutamate concentration went high, and <i>tetA</i> of PopQC was upregulated to express. The results suggested that the system can help individuals with higher intracellular glutamate concentration express more TetA, so that be able to survive in the tetracycline condition.</p> |
| − | <div class="panel-group" id="results">
| + | </div> |
| − | <div class="panel panel-default" style="margin-right: 10%;margin-left: 10%;">
| + | |
| − | <div class="panel-heading panelheadingcursor" data-toggle="collapse" data-parent="#results" data-target="#collapse9" style="background-color: #A098B6;">
| + | |
| − | <h4 class="panel-title" style="height: 35px;">
| + | |
| − | <a style="font-size: 25px;">OD<sub>600</sub> Reference Point</a> | + | |
| − | </h4>
| + | |
| − | </div>
| + | |
| − | <div id="collapse9" class="panel-collapse collapse">
| + | |
| − | | + | |
| − | <img src="https://static.igem.org/mediawiki/2018/6/60/T--NKU_CHINA--OD600Reference_point.png" class="img-responsive center-block">
| + | |
| − | <p style=" font-size: 20px;line-height: 25px;margin-left: 10px;color: #000"> According to the Reference OD< sub> 600</ sub>, We calculate the final result: < br> | + | |
| − | OD<sub>600</sub>/Abs<sub>600</sub>=3.818 <br>
| + | |
| − | All cell density readings using this instrument with the same settings and volume can be converted to OD<sub>600</sub>, so that we can use this ratio to convert subsequent experimental data.
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | <div class="panel panel-default" style="margin-right: 10%;margin-left: 10%;">
| + | |
| − | <div class="panel-heading panelheadingcursor" data-toggle="collapse" data-parent="#results" data-target="#collapse10" style="background-color: #B7B1C8;">
| + | |
| − | <h4 class="panel-title" style="height: 35px;">
| + | |
| − | <a style="font-size: 25px;">Particle Standard Curve</a>
| + | |
| − | </h4>
| + | |
| − | </div>
| + | |
| − | <div id="collapse10" class="panel-collapse collapse">
| + | |
| − | <div class="panel-body">
| + | |
| − | <img src="https://static.igem.org/mediawiki/2018/3/35/T--NKU_CHINA--ParticleStandardCurve_list.png" class="img-responsive center-block">
| + | |
| − | <p style="font-size: 20px;line-height: 25px;margin-left: 10px;color: #000">We prepare a dilution series of monodisperse silica microspheres and measure the Abs<sub>600</sub> in plate reader. The size and optical characteristics of these microspheres are similar to cells, and there is a known amount of particles per volume. This measurement allows us to construct a standard curve of particle concentration which can be used to convert Abs<sub>600</sub> measurements to an estimated number of cells.
| + | |
| − | </p>
| + | |
| − | <img src="https://static.igem.org/mediawiki/2018/5/56/T--NKU_CHINA--Curve_Logscale.png" class="img-responsive center-block">
| + | |
| − | <p style="font-size: 20px;line-height: 25px;margin-left: 10px;color: #000">The final result:<br>mean of med-high levels=6.24E+08</p>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | <div class="panel panel-default" style="margin-right: 10%;margin-left: 10%;">
| + | |
| − | <div class="panel-heading panelheadingcursor" data-toggle="collapse" data-parent="#results" data-target="#collapse11" style="background-color: #D1CDDC;">
| + | |
| − | <h4 class="panel-title" style="height: 35px;">
| + | |
| − | <a style="font-size: 25px;">Fluorescein Standard Curve</a>
| + | |
| − | </h4>
| + | |
| − | </div>
| + | |
| − | <div id="collapse11" class="panel-collapse collapse">
| + | |
| − | <div class="panel-body">
| + | |
| − | <p style="font-size: 20px;line-height: 25px;margin-left: 10px;color: #000"> We prepare a dilution series of fluorescein in four replicates and measure the fluorescence in a 96 well plate in plate reader. By measuring these we generate a standard curve of fluorescence for fluorescein concentration. We will be able to use this to convert our cell based readings to an equivalent fluorescein concentration.
| + | |
| − | </p>
| + | |
| − | <img src="https://static.igem.org/mediawiki/2018/d/d1/T--NKU_CHINA--FluoresceinStandardcurve_list.png" class="img-responsive center-block">
| + | |
| − | <img src="https://static.igem.org/mediawiki/2018/0/00/T--NKU_CHINA--FluoresceinStandardCurve_logscale.png" class="img-responsive center-block">
| + | |
| − | <p style="font-size: 20px;line-height: 25px;margin-left: 10px;color: #000"> Final results:<br>Mean µM fluorescein / a.u.=3.60E-05<br>MEFL / a.u.=2.17E+08</p>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | <div class="panel panel-default" style="margin-right: 10%;margin-left: 10%;">
| + | |
| − | <div class="panel-heading panelheadingcursor" data-toggle="collapse" data-parent="#results" data-target="#collapse12" style="background-color: #EAE8EF;">
| + | |
| − | <h4 class="panel-title" style="height: 35px;">
| + | |
| − | <a style="font-size: 25px;">Raw Plate Reader Measurements</a>
| + | |
| − | </h4>
| + | |
| − | </div>
| + | |
| − | <div id="collapse12" class="panel-collapse collapse">
| + | |
| − | <div class="panel-body">
| + | |
| − | <img src="https://static.igem.org/mediawiki/2018/3/39/T--NKU_CHINA--RawPlateReader_Measurements1.png" class="img-responsive center-block">
| + | |
| − | <hr>
| + | |
| − | <img src="https://static.igem.org/mediawiki/2018/2/28/T--NKU_CHINA--RawPlateReader_Measurements2.png" class="img-responsive center-block">
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | </div>
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| − | </main>
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| − | <footer>
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| − | <div class="container-fluid myFooter">
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| − | <h3 style="color: white; font-size: 30px;">Contacts</h3>
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| − | Nankai University</br>
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| − | No.94 Weijin Road, Nankai District</br>
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| − | Tianjin, P.R.China 300071</br>
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| − | <p class="Copyright" style="text-align: center; color: white;">Copyright © Team NKU_CHINA 2018</p>
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| − | </html> | + | <p class="tuzhu"><strong>Figure 2. The relationship of extra- and intra-cellular glutamate concentration and FI of GFP /OD<sub>600</sub> in plateau stage.</strong> The strains were cultured in fermentation medium with 10 µg/mL tetracycline for 24 hours. Intracellular glutamate concentration, OD<sub>600</sub> and FI of GFP were measured in plateau stage. Data indicate mean values ± standard deviations from three independent experiments performed in triplicates. <strong>a. The relationship between extra- and intracellular glutamate concentration.</strong> The value illustrates the relationship between glutamate concentration in medium and intracellular glutamate concentration. <strong>b. The relationship between intracellular glutamate concentration and FI of GFP /OD<sub>600</sub> in Plateau stage.</strong> The value illustrates the effect of glutamate concentration on GFP fluorescence intensity, which is normalized against OD<sub>600</sub>. *Significantly different (P < 0.05) by Student's t-test.</p> |
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