Difference between revisions of "Team:ShanghaiFLS China/Experiments"

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<title>ShanghaiFLS_China: Experiment</title>
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<h1>Experiments</h1>
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<p>Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.</p>
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            <h1>
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            <a href="https://2019.igem.org/Team:ShanghaiFLS_China">Team:ShanghaiFLS_China</a>
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        <label for="drop" class="toggle"></label>
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                <li class="mr-lg-4 mr-3"><a href="https://2019.igem.org/Team:ShanghaiFLS_China">Home</a></li>
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                <li class="mr-lg-4 mr-3">
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                <label for="drop-2" class="toggle">Team<span class="fa fa-angle-down" aria-hidden="true"></span> </label>
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                <a>Team<span class="fa fa-angle-down" aria-hidden="true"></span></a>
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                <ul>
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                    <li><a href="https://2019.igem.org/Team:ShanghaiFLS_China/Team">Team</a></li>
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                    <li><a href="https://2019.igem.org/Team:ShanghaiFLS_China/Collaborations">Collaborations</a></li>
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                </ul>
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                </li>
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                <li class="mr-lg-4 mr-3 active">
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                <label for="drop-2" class="toggle">Project<span class="fa fa-angle-down" aria-hidden="true"></span> </label>
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                <ul>
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                    <li><a href="https://2019.igem.org/Team:ShanghaiFLS_China/Description">Description</a></li>
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                    <li><a href="https://2019.igem.org/Team:ShanghaiFLS_China/Design">Design</a></li>
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                    <li><a href="https://2019.igem.org/Team:ShanghaiFLS_China/Experiments">Experiments</a></li>
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                    <li><a href="https://2019.igem.org/Team:ShanghaiFLS_China/Notebook">Notebook</a></li>
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                    <li><a href="https://2019.igem.org/Team:ShanghaiFLS_China/Contribution">Contribution</a></li>
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                    <li><a href="https://2019.igem.org/Team:ShanghaiFLS_China/Results">Results</a></li>
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                    <li><a href="https://2019.igem.org/Team:ShanghaiFLS_China/Model">Model</a></li>
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                    <li><a href="https://2019.igem.org/Team:ShanghaiFLS_China/Demonstrate">Demonstrate</a></li>
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                    <li><a href="https://2019.igem.org/Team:ShanghaiFLS_China/Attributions">Attributions</a></li>
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                </ul>
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                </li>
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                <li class="mr-lg-4 mr-3">
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                <label for="drop-2" class="toggle">Parts<span class="fa fa-angle-down" aria-hidden="true"></span> </label>
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                <a>Parts<span class="fa fa-angle-down" aria-hidden="true"></span></a>
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                <input type="checkbox" id="drop-2"/>
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                <ul>
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                    <li><a href="https://2019.igem.org/Team:ShanghaiFLS_China/Parts">Parts Overview</a></li>
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                    <li><a href="https://2019.igem.org/Team:ShanghaiFLS_China/Basic_Part">Basic Parts</a></li>
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                    <li><a href="https://2019.igem.org/Team:ShanghaiFLS_China/Composite_Part">Composite Parts</a></li>
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                    <li><a href="https://2019.igem.org/Team:ShanghaiFLS_China/Part_Collection">Part Collection</a></li>
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                </ul>
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                </li>
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                <li class="mr-lg-4 mr-3"><a href="https://2019.igem.org/Team:ShanghaiFLS_China/Safety">Safety</a></li>
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                <label for="drop-2" class="toggle">Human Practices<span class="fa fa-angle-down" aria-hidden="true"></span> </label>
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                <ul>
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                    <li><a href="https://2019.igem.org/Team:ShanghaiFLS_China/Human_Practices">Integrated Human Practices</a></li>
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                    <li><a href="https://2019.igem.org/Team:ShanghaiFLS_China/Public_Engagement">Education and Engagement</a></li>
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                </ul>
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                </li>
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                <div class="col-md-8 w3layouts_banner_margin">
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                    <h2 class="editContent">Experiment</h2>
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                </div>
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</section>
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<section class="about py-5" id="about">  
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Please remember to put all characterization and measurement data for your parts on the corresponding Registry part pages.  
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    <div class="container py-3">
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        <div class="row about-grids">
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                <p class="my-4">i. The following data were acquired after 108 h 50 mL YNM (1.34% yeast nitrogenous base with 0.5% methanol, 50 µg/mL histidine and 40 µg/mL biotin) incubation, sampled every 12 h. The starting concentration was 1 OD600.<br /><br />ii. 0.5% Methanol was added to the media every 24 hours to maintain the methanol concentration.<br /><br />iii. A short-term methanol induction experiment was performed after the 108 h incubation to characterize the instant response to methanol induction after the yeast cells are accustomed to methanol media, hence indicating the overall induction activity of the construct. The yeast cells were collected, rinsed and stored at 4 ˚C overnight to allow GFP to fully degrade. The strains were then incubated in higher-concentration 50 mL YNM media (1.34% yeast nitrogenous base with 0.75% methanol, 50 µg/mL histidine and 40 µg/mL biotin) for 12 hours and sampled every 2 hours. The starting concentration was 3 OD600.<br /><br />iv. For each experimental construct, two identical strains were created (labeled 1 and  2) and three parallels were performed for each strain.<br /><br />v. A Biotek Synergy 2 plate reader was used to measure the GFP mean fluorescence intensities and the OD600 absorbance of our samples.<br /><br />vi. Methanol concentration measurements were performed with a Shenzhen Sieman M100 Biosensors Analyzer.<br /><br />vii. OD600 is used as an indicator of yeast concentration in the experiment.<br /><br />viii. Measured unit cell GFP production is the ratio of the GFP mean fluorescence intensities to the OD600 absorbance measured by the plate reader. It shall serve as an indicator of the expression level of GFP at the sampled time point.<br /><br />ix. Aggregate unit cell GFP production is the calculated total unit cell GFP fluorescence intensity. It accounts for the GFP that has degraded over the incubation period to more accurately reflect the aggregate production rate.<br /><br />x. Total GFP production is the product of the yeast concentration and the aggregate unit cell GFP. It reflects the total GFP production of the reaction system of the strain.<br /><br />xi. Total GFP production per gram methanol is the ratio of the total GFP production to the total methanol consumption. It reflects the overall conversion rate of methanol to the product.
 
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    <div class="container">
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            <div class="w3layouts-grid-left w3layouts-grid-right text-center">  
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<h3>What should this page contain?</h3>
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                <div class="social mb-4">  
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<ul>
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                    <ul>  
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<li> Protocols </li>
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                        <li><a><span class="fa qmr-2 fa-twitter"></span></a><a>igem2019_sfls</a></li>
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<li> Experiments </li>
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                        <li><a><span class="fa qmr-2 fa-instagram"></span></a><a>igem_shanghaifls_china</a></li>
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<li> Documentation of the development of your project </li>
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                    </ul>  
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</ul>
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                </div>  
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            </div>  
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        <div class="copyright text-center">
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            <p>Copyright &copy; iGEM 2019, team: ShanghaiFLS_China. All rights reserved. Email: SFLS_x_iGEM@outlook.com</p>
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<div class="highlight decoration_A_full">
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            <a href="#" class="move-top scroll"><span class="fa fa-angle-up" aria-hidden="true"></span></a>  
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<h3>Inspiration</h3>
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        </div>  
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<ul>
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    </div>  
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<li><a href="https://2014.igem.org/Team:Colombia/Protocols">2014 Colombia </a></li>
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</footer>  
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<li><a href="https://2014.igem.org/Team:Imperial/Protocols">2014 Imperial </a></li>
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<li><a href="https://2014.igem.org/Team:Caltech/Project/Experiments">2014 Caltech </a></li>
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</ul>
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Revision as of 09:55, 18 October 2019

ShanghaiFLS_China: Experiment

i. The following data were acquired after 108 h 50 mL YNM (1.34% yeast nitrogenous base with 0.5% methanol, 50 µg/mL histidine and 40 µg/mL biotin) incubation, sampled every 12 h. The starting concentration was 1 OD600.

ii. 0.5% Methanol was added to the media every 24 hours to maintain the methanol concentration.

iii. A short-term methanol induction experiment was performed after the 108 h incubation to characterize the instant response to methanol induction after the yeast cells are accustomed to methanol media, hence indicating the overall induction activity of the construct. The yeast cells were collected, rinsed and stored at 4 ˚C overnight to allow GFP to fully degrade. The strains were then incubated in higher-concentration 50 mL YNM media (1.34% yeast nitrogenous base with 0.75% methanol, 50 µg/mL histidine and 40 µg/mL biotin) for 12 hours and sampled every 2 hours. The starting concentration was 3 OD600.

iv. For each experimental construct, two identical strains were created (labeled 1 and 2) and three parallels were performed for each strain.

v. A Biotek Synergy 2 plate reader was used to measure the GFP mean fluorescence intensities and the OD600 absorbance of our samples.

vi. Methanol concentration measurements were performed with a Shenzhen Sieman M100 Biosensors Analyzer.

vii. OD600 is used as an indicator of yeast concentration in the experiment.

viii. Measured unit cell GFP production is the ratio of the GFP mean fluorescence intensities to the OD600 absorbance measured by the plate reader. It shall serve as an indicator of the expression level of GFP at the sampled time point.

ix. Aggregate unit cell GFP production is the calculated total unit cell GFP fluorescence intensity. It accounts for the GFP that has degraded over the incubation period to more accurately reflect the aggregate production rate.

x. Total GFP production is the product of the yeast concentration and the aggregate unit cell GFP. It reflects the total GFP production of the reaction system of the strain.

xi. Total GFP production per gram methanol is the ratio of the total GFP production to the total methanol consumption. It reflects the overall conversion rate of methanol to the product.