Difference between revisions of "Team:Tokyo Tech/InterLab"

 
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<section class="engine"><a href="https://mobirise.info/k">how to develop your own website</a></section><section class="header10 cid-r5UmXXtySa mbr-fullscreen mbr-parallax-background" id="header10-1f">
  
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<section class="engine"><a href="https://mobirise.info/j">website templates</a></section><section class="cid-r5ehuhHDnG mbr-fullscreen mbr-parallax-background" id="header2-6">
 
  
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                <h1 class="mbr-section-title mbr-bold pb-3 mbr-fonts-style display-1">PROJECT</h1>
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            <h1 class="mbr-section-title align-right mbr-bold pb-3 mbr-fonts-style display-1">InterLab</h1>
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            <h3 class="mbr-text align-right pb-3 mbr-fonts-style display-5">We introduced eight plasmids (2 controls and 6 test devices) to E.coli DH5α by following the protocol and then evaluate the measurement by measuring its OD600. We used TECAN infinite M200 PRO as a plate reader.</h3>
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                    <a class="btn btn-md btn-secondary display-4" href="https://mobirise.com">LEARN MORE</a>
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                    <a class="btn btn-md btn-white-outline display-4" href="https://mobirise.com">LIVE DEMO</a>
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              <div class="section-text align-center mbr-white mbr-fonts-style display-5">"Not only in the synthetic biology, but in the general scientific field, it is definitely important to get the result with high reproducibility and high credibility."</div><br>
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              <div class="section-text align-center mbr-white mbr-fonts-style display-5">"To achieve this, in iGEM, InterLab Study has been conducted by collecting and comparing the result of GFP measurements with a plate reader."</div><br>
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              <div class="section-text align-center mbr-white mbr-fonts-style display-5"><a href="https://static.igem.org/mediawiki/2018/0/09/2018_InterLab_Plate_Reader_Protocol.pdf" target="_blank">--- iGEM Headquarters</a></div></div>
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                    <h1 class="card-title py-3 mbr-fonts-style display-7">Transformation</h1>
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                            <p class="mbr-text mb-0 mbr-fonts-style display-7"><br>Dengue virus, which is in the flavivirus family, is a worldwide spread virus and has huge impact on society, however, not many developing countries are recognizing its danger.<br><br></p>
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                    <h2 class="mbr-text mbr-fonts-style display-7">We introduced eight plasmids from the Distribution Kit to E.Coli DH5α.&nbsp;</h2>
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                    <h3 class="card-title py-3 mbr-fonts-style display-7">Calibration
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                    <h4 class="mbr-text mbr-fonts-style display-7">We conducted three tests and calibrated the machine.</h4>
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                    <h2 class="card-title py-3 mbr-fonts-style display-7">Cell Measurement
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                    <h3 class="mbr-text mbr-fonts-style display-7">We measured the OD600 and the fluorescence of the transformed cells.&nbsp;</h3>
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                    <h3 class="card-title py-3 mbr-fonts-style display-7">CFU</h3>
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                    <h4 class="mbr-text mbr-fonts-style display-7">
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                      We measured the  Colony Forming Units&nbsp;using two types of transformation cells.</h4>
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             <div class="mbr-text col-12 col-md-8 mbr-fonts-style display-7">Dengue virus is unique in terms of its four different serotypes. Multiple infection can easily cause severe dengue, appearing hemorrhage and organ damage. It is important to grasp which serotype the patient is infected, however, there is not enough data about each serotype in a year.<div><br></div><div>To tackle the situation, we took the following two approaches.</div></div>
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                <h2 class="mbr-text testimonial-text mbr-fonts-style display-5"><strong>Transformation</strong><br></h2>
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                <h3 class="mbr-author-name pt-4 mb-2 mbr-fonts-style display-7"><span style="font-weight: normal;">
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                    We took the following eight plasmids from the Distribution Kit and introduced them to E.Coli DH5α. Then, we selected two colonies and cultured them for 16 hours.
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                </span></h3>
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                <p class="mbr-author-desc mbr-fonts-style display-7"><br><br></p>
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              <img src="https://static.igem.org/mediawiki/2018/1/11/T--Tokyo_Tech--Transformation.png" alt="" title="">
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                <ol>
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                <h2 class="mbr-text testimonial-text mbr-fonts-style display-5"><strong>Calibration</strong></h2>
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                    <li><span style="font-size: 1rem;"><strong>MODELLING</strong>&nbsp;- We succeeded in the development of the serotype prediction system using stochastic process analysis. This system can predict the patient's serotype by simulating the past data. </span><a href="https://mobirise.co/" style="font-size: 1rem; background-color: rgb(255, 255, 255);">Try it now!</a><br></li>
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                <h3 class="mbr-author-name pt-4 mb-2 mbr-fonts-style display-7"><span style="font-weight: normal;">We conducted the following three measurements to get the calibration values as a preparation for the Cell Measurement.
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                    <li><strong>FINDING FLAVI</strong>&nbsp;- We also developed the simple and fast testing kit that can detect serotype with fluorescence, so that we can check the patient easily and get enough data to estimate the patients’ serotypes more accurate. <a href="https://mobirise.co/">Try it now!</a></li>
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                <h3 class="mbr-author-name pt-4 mb-2 mbr-fonts-style display-7"><span style="font-weight: normal;">①Measurment of the LUDOX CL-X solution to obtain a conversation factor to transform the  absorbance data into a comparable OD600 measurement.</span></h3>
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                <h3 class="mbr-author-name pt-4 mb-2 mbr-fonts-style display-7"><span style="font-weight: normal;">②ABS600 measument of the dilution series  of silica beads to construct a standard curve of particle concentration which can be used to convert Abs 600 measurements to an estimated number of cells.&nbsp;</span></h3>
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                <h3 class="mbr-author-name pt-4 mb-2 mbr-fonts-style display-7"><span style="font-weight: normal;">③Fluorescence measurement of the dilution series of Fluorescein to generate a standard curve of fluorescence for fluorescein concentration. You will be able to use this to convert your cell based readings to an equivalent fluorescein concentration.&nbsp;</span></h3>
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                <h3 class="mbr-author-name pt-4 mb-2 mbr-fonts-style display-7"><span style="font-weight: normal;"></span></h3>
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                <p class="mbr-author-desc mbr-fonts-style display-7"><br><br></p>
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              <img src="https://static.igem.org/mediawiki/2018/7/73/T--Tokyo_Tech--Calibration.png" alt="" title="">
 
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             <div class="mbr-text col-12 col-md-8 mbr-fonts-style display-7">In the future, this system can contribute to other flavivirus detection system.<div><strong><br></strong></div></div>
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                <blockquote><h3><strong>Discussion about Calibration</strong></h3>
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                  <h3>From the Experimental results,Particles / Abs600 = 5.18*10⁸ and MEFL / a.u. = 1.24*10⁹ was found.</h3></blockquote>
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                    <a class="btn btn-primary display-4" href="https://mobirise.co">LEARN MORE</a>
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                  Cell Measurement
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                    <a class="btn btn-black-outline display-4" href="https://mobirise.co">LIVE DEMO</a>
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                </strong></h2>
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                <h3 class="mbr-author-name pt-4 mb-2 mbr-fonts-style display-7"><span style="font-weight: normal;">We measured the OD600 and the fluorescence of the transformed cells in zero and six hours later. Then we evaluated the measurement data by Calibration values.</span>&nbsp;<br></h3>
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                <blockquote><h3> <strong>Discussion about Cell Mesurement</strong></h3><h3>The results show the changes in optical density (λ = 600 nm) of the transformed cells. They show that the cells grew steadily.
 +
</h3><h3>However, when we measured the fluorescence, some values appeared negative or go down after 6-hour culturing. Thus, the Fluorescence/OD and MEFL/Particle differed from the theoretical values.
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                <h2 class="mbr-text testimonial-text mbr-fonts-style display-5"><strong>CFU</strong></p>
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                <h3 class="mbr-author-name pt-4 mb-2 mbr-fonts-style display-7"><span style="font-weight: normal;">We diluted both positive and negative controls so that their OD600 became 0.1. Then, we prepared dilution series of them and spread 8.0*10³, 8.0*10⁴, 8.0*10⁵ to the agar medium, counted the number of colonies after 18 hours of cultivation.&nbsp;</span><br></h3>
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              <img src="https://static.igem.org/mediawiki/2018/2/23/T--Tokyo_Tech--CFU.png" alt="" title="">
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                <blockquote><h2><strong>Discussion about CFU</strong> </h2><h3>We counted the CFU (Colony Forming Unit) and the following figures show the number of colony on each plate. Then, we calculated the CFU / mL in the starting sample when the optical density (λ = 600 nm) is 0.1.
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</h3></blockquote>
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            <div class="mbr-text col-12 col-md-8 mbr-fonts-style display-5"><h2><strong>Reference
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</strong></h2><p></p><div><strong>　<a href="https://2018.igem.org/Measurement/InterLab">https://2018.igem.org/Measurement/InterLab
 +
</a></strong></div><div><strong>　<a href="https://2017.igem.org/Team:William_and_Mary/Interlab">https://2017.igem.org/Team:William_and_Mary/Interlab
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</a></strong></div><div><strong>
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</strong></div><div><strong><br></strong></div><p></p></div>
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Latest revision as of 03:12, 18 October 2018

<!DOCTYPE html>

InterLab
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InterLab

We introduced eight plasmids (2 controls and 6 test devices) to E.coli DH5α by following the protocol and then evaluate the measurement by measuring its OD600. We used TECAN infinite M200 PRO as a plate reader.

"Not only in the synthetic biology, but in the general scientific field, it is definitely important to get the result with high reproducibility and high credibility."

"To achieve this, in iGEM, InterLab Study has been conducted by collecting and comparing the result of GFP measurements with a plate reader."

--- iGEM Headquarters

Transformation

We introduced eight plasmids from the Distribution Kit to E.Coli DH5α. 

Calibration

We conducted three tests and calibrated the machine.

Cell Measurement

We measured the OD600 and the fluorescence of the transformed cells. 

CFU

We measured the Colony Forming Units using two types of transformation cells.

Transformation

We took the following eight plasmids from the Distribution Kit and introduced them to E.Coli DH5α. Then, we selected two colonies and cultured them for 16 hours.



Calibration

We conducted the following three measurements to get the calibration values as a preparation for the Cell Measurement.

①Measurment of the LUDOX CL-X solution to obtain a conversation factor to transform the absorbance data into a comparable OD600 measurement.

②ABS600 measument of the dilution series of silica beads to construct a standard curve of particle concentration which can be used to convert Abs 600 measurements to an estimated number of cells. 

③Fluorescence measurement of the dilution series of Fluorescein to generate a standard curve of fluorescence for fluorescein concentration. You will be able to use this to convert your cell based readings to an equivalent fluorescein concentration. 



Discussion about Calibration

From the Experimental results,Particles / Abs600 = 5.18*10⁸ and MEFL / a.u. = 1.24*10⁹ was found.

Cell Measurement

We measured the OD600 and the fluorescence of the transformed cells in zero and six hours later. Then we evaluated the measurement data by Calibration values. 

Discussion about Cell Mesurement

The results show the changes in optical density (λ = 600 nm) of the transformed cells. They show that the cells grew steadily.

However, when we measured the fluorescence, some values appeared negative or go down after 6-hour culturing. Thus, the Fluorescence/OD and MEFL/Particle differed from the theoretical values.

CFU

We diluted both positive and negative controls so that their OD600 became 0.1. Then, we prepared dilution series of them and spread 8.0*10³, 8.0*10⁴, 8.0*10⁵ to the agar medium, counted the number of colonies after 18 hours of cultivation. 

Discussion about CFU

We counted the CFU (Colony Forming Unit) and the following figures show the number of colony on each plate. Then, we calculated the CFU / mL in the starting sample when the optical density (λ = 600 nm) is 0.1.

Reference


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