Difference between revisions of "Team:Alverno CA/InterLab"

 
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    <title>Alverno iGEM 2016</title>
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<h1><center>Alverno iGEM Interlab Study</center></h1>
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<h1><center>Quantifying different sources of variation in gene expression using  
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fluorescent transformed E. coli bacteria</center></h1>
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<center><img src="https://s-media-cache-ak0.pinimg.com/originals/86/08/18/860818cfc65e5ff04725bb0f0c05a8af.png" alt="Alverno iGEM Logo" style="width:300px;"></center>
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    <br>
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    <center>
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        <h2>Interlab Study</h2>
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    </center>
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    <center>
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        <h3>Quantifying different sources of variation in gene expression<br> using
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        fluorescent transformed E. coli bacteria</h3>
 +
    </center>
 +
 
 +
 
 +
    <div class="demo" id="container">
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        <center>
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            <ul>
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                <li style="list-style: none"><br>
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                <br>
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                <li><img src=
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                "https://static.igem.org/mediawiki/2016/6/6c/T--Alverno_CA--Interlab1.jpg"
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                style="height:215px;width:215px;" title=
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                "Growing bacteria in LB broth">
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                </li>
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                <li><img src=
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                "https://static.igem.org/mediawiki/2016/9/9a/T--Alverno_CA--Interlab2.jpg"
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                style="height:215px;width:215px;" title=
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                "Looking at plates">
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                </li>
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                <li><img src=
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                "https://static.igem.org/mediawiki/2016/2/25/T--Alverno_CA--Interlab3.jpg"
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                style="height:215px;width:215px;" title=
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                "Analyzing data">
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                <li><img src=
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                "https://static.igem.org/mediawiki/2016/f/f5/T--Alverno_CA--Interlab4.jpg"
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                style="height:215px;width:215px;" title=
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                "Pipetting bacteria to be grown">
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                <li><img src=
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                "https://static.igem.org/mediawiki/2016/8/83/T--Alverno_CA--Interlab5.jpg"
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                style="height:215px;width:215px;" title=
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                "Analyzing plate reader data">
 +
                </li>
 +
 
 +
 
 +
                <li><img src=
 +
                "https://static.igem.org/mediawiki/2016/e/e8/T--Alverno_CA--Interlab6.jpg"
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                style="height:215px;width:215px;" title=
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                "Our fluorescent bacteria <br>grown in LB broth">
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                </li>
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 +
 
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                <li><img src=
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                "https://static.igem.org/mediawiki/2016/8/83/T--Alverno_CA--Interlab7.jpg"
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                style="height:215px;width:215px;" title=
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                "Diluting cultures">
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                <li><img src=
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                "https://static.igem.org/mediawiki/2016/4/49/T--Alverno_CA--Interlab8.jpg"
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                style="height:215px;width:215px;" title=
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                "Pipetting FITC standard curve">
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                <li><img src=
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                "https://static.igem.org/mediawiki/2016/8/8c/T--Alverno_CA--Interlab9.jpg"
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                style="height:215px;width:215px;" title=
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                "Plate reader scan for ODs">
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 +
    <h5>
 +
    </h5>
 +
 
 +
 
 +
    <h4>Summary:</h4>
 +
    <h5></h5>
 +
 
 +
    <h5>As part of the 2016 iGEM Interlab Study, we tested 3 different
 +
    fluorescent protein expression plasmids, J23101, J23106, and J23117 with
 +
    positive and negative controls. We transformed the parts with iGEM's
 +
    transformation protocol. Our bacteria were cultured on a LB agar plate and
 +
    cultured in LB broth. To measure the differences in fluorescence expressed
 +
    by different strength plasmids, we used a plate reader with an OD600
 +
    reference point and FITC standard curve.</h5>
 +
 
 +
 
 +
    <h5>
 +
    </h5>
 +
<br><br>
 +
 
 +
    <h4>Project Description:</h4>
 +
    <h5></h5>
 +
 
 +
    <h5>The 2016 iGEM Interlab Study aims to compare results obtained by various
 +
    teams in order to quantify the expression of 5 different constructs using
 +
    fluorescence, which provides a useful insight into expression levels that
 +
    can be monitored without disrupting cells. Each device used in the study is
 +
    in the pSB1C3 plasmid backbone and are fluorescent protein expression
 +
    plasmids of different strength promoters. The devices we tested were
 +
    J23101, J23106, J23117 (Test Devices 1, 2, and 3). We used positive control
 +
    I20270 and negative control R0040. J23101, with a strong promoter, showed
 +
    the highest amount of fluorescence. J23117, with a weak promoter, showed
 +
    the lowest amount of fluorescence, and J23106, with the medium strength
 +
    promoter, showed fluorescence amounts between the other two. Our test
 +
    devices were inserted into DH5alpha E. coli, which were transformed
 +
    according to the iGEM transformation protocol. Our transformed cells were
 +
    plated on LB plates with chloramphenicol and incubated overnight at 37° C.
 +
    Two colonies were picked from each plate (with the exception of Test Device
 +
    3, with which there was only one) and were inoculated in 15mL test tubes.
 +
    These devices were diluted, and then measured in a plate reader with an
 +
    excitation wavelength of 35nm, according to iGEM’s measurement
 +
    protocol.</h5>
 +
 
 +
 
 +
    <h5>
 +
    </h5>
 +
 
 +
 
 +
    <h5>
 +
    </h5>
 +
<br><br>
 +
 
 +
    <h4>Protocol:</h4>
 +
    <h5></h5>
 +
 
 +
    <h5><a href=
 +
    "https://static.igem.org/mediawiki/2016/c/c5/InterLab_iGEM2016_Plate_Reader_Protocol_Updated_July.pdf">
 +
    Plate Reader Protocol</a>
 +
    </h5>
 +
 
 +
 
 +
    <h5><a href=
 +
    "https://static.igem.org/mediawiki/parts/6/67/IGEM_Registry_-_Transformation_Protocol.pdf">
 +
    Transformation Protocol</a>
 +
    </h5>
 +
 
  
−
<p><h3>Summary:</h3> </p>
+
    <h5>
−
<p>Leverage agile frameworks to provide a robust synopsis for high level overviews. Iterative approaches to corporate strategy foster collaborative thinking to further the overall value proposition. Organically grow the holistic world view of disruptive innovation via workplace diversity and empowerment.</p>  
+
    </h5>
  
−
<p><h3>Project Description:</h3></p>
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<br><br>
−
<p>We tested 3 different fluorescent protein expression plasmids, J23101, J23106, and J23117. We transformed the parts with iGEM's transformation protocol. Our bacteria was cultured on a LB agar plate and cultured in LB broth. To measure the differences in fluorescence caused by each plasmid, we used a plate reader with an OD600 reference point and FITC standard curve.<p>
+
    <h4>Results:</h4>
 +
<h5>You can download our data sheet <a href="https://goo.gl/uofZIO">here.</a></h5>
  
−
<p><h3>Protocol:</h3> </p>
+
<img class:icons src="https://static.igem.org/mediawiki/2016/0/00/T--Alverno_CA--interabs.png" alt="Abs600" style="width:800px;">
−
<p><a href="https://static.igem.org/mediawiki/2016/c/c5/InterLab_iGEM2016_Plate_Reader_Protocol_Updated_July.pdf">Plate Reader Protocol</a></p>  
+
<h5>Graph of Absorbance (600) over a 6 hour time period.
−
<p><a href="https://static.igem.org/mediawiki/parts/6/67/IGEM_Registry_-_Transformation_Protocol.pdf">Transformation Protocol</a></p>  
+
</h5>
 +
<br><br>
 +
<img class:icons src="https://static.igem.org/mediawiki/2016/c/ca/T--Alverno_CA--interfl.png" alt="Fl" style="width:800px;">
 +
<h5>Graph of Fluorescence in AU over a 6 hour time period.
 +
</h5>
 +
<br><br>
 +
<img class:icons src="https://static.igem.org/mediawiki/2016/8/82/T--Alverno_CA--interflabs2.png" alt="Fl/Abs" style="width:800px;">
 +
<h5>Graph of Fluorescence/Absorbance in AU (log scale) over a 6 hour time period. Amounts of Fl/Abs600 are consistent with the strengths of the promoters.
 +
</h5>
 +
<br><br>
 +
<img class:icons src="https://static.igem.org/mediawiki/2016/c/c2/T--Alverno_CA--interfitc.png" alt="FITC" style="width:800px;">
 +
<h5>FITC Fluorescence standard curve. Row A had pipetting errors and so is different from the other curves.
 +
</h5>
 +
<br>
 +
 
  
−
<p><h3>Results:</h3></p>
 
−
<center><img src="http://placehold.it/350" alt="Placeholder Image" style="width:300px;"><img src="http://placehold.it/350" alt="Placeholder Image" style="width:300px;"><img src="http://placehold.it/350" alt="Placeholder Image" style="width:300px;"></center>
 
  
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Latest revision as of 03:32, 20 October 2016

Alverno iGEM 2016


Interlab Study

Quantifying different sources of variation in gene expression
using fluorescent transformed E. coli bacteria































Summary:

As part of the 2016 iGEM Interlab Study, we tested 3 different fluorescent protein expression plasmids, J23101, J23106, and J23117 with positive and negative controls. We transformed the parts with iGEM's transformation protocol. Our bacteria were cultured on a LB agar plate and cultured in LB broth. To measure the differences in fluorescence expressed by different strength plasmids, we used a plate reader with an OD600 reference point and FITC standard curve.


Project Description:

The 2016 iGEM Interlab Study aims to compare results obtained by various teams in order to quantify the expression of 5 different constructs using fluorescence, which provides a useful insight into expression levels that can be monitored without disrupting cells. Each device used in the study is in the pSB1C3 plasmid backbone and are fluorescent protein expression plasmids of different strength promoters. The devices we tested were J23101, J23106, J23117 (Test Devices 1, 2, and 3). We used positive control I20270 and negative control R0040. J23101, with a strong promoter, showed the highest amount of fluorescence. J23117, with a weak promoter, showed the lowest amount of fluorescence, and J23106, with the medium strength promoter, showed fluorescence amounts between the other two. Our test devices were inserted into DH5alpha E. coli, which were transformed according to the iGEM transformation protocol. Our transformed cells were plated on LB plates with chloramphenicol and incubated overnight at 37° C. Two colonies were picked from each plate (with the exception of Test Device 3, with which there was only one) and were inoculated in 15mL test tubes. These devices were diluted, and then measured in a plate reader with an excitation wavelength of 35nm, according to iGEM’s measurement protocol.


Protocol:

Plate Reader Protocol
Transformation Protocol


Results:

You can download our data sheet here.
Abs600
Graph of Absorbance (600) over a 6 hour time period.


Fl
Graph of Fluorescence in AU over a 6 hour time period.


Fl/Abs
Graph of Fluorescence/Absorbance in AU (log scale) over a 6 hour time period. Amounts of Fl/Abs600 are consistent with the strengths of the promoters.


FITC
FITC Fluorescence standard curve. Row A had pipetting errors and so is different from the other curves.