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− | <center><img src="https://static.igem.org/mediawiki/2018/ | + | <center><img src="https://static.igem.org/mediawiki/2018/3/34/T--Tec-Chihuahua--interlabletter.png" width="55%" height="30%"/></center><h1></h1><h1></h1><h2></h2><h4></h4> |
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<p align="justify">All of the 2018 iGEM teams were invited to participate in the Fifth International InterLaboratory Measurement Study in synthetic biology. Team Tec-Chihuahua is excited about working on an international and collaborative experiment!</p> | <p align="justify">All of the 2018 iGEM teams were invited to participate in the Fifth International InterLaboratory Measurement Study in synthetic biology. Team Tec-Chihuahua is excited about working on an international and collaborative experiment!</p> | ||
− | + | <div class="a"><p align="justify">The protocols were actually very simple to understand and we didn't have any problems following them. Without a doubt, it was an enlightening experience full of challenges that helped us improve laboratory work during the development of the project. When we started working on it, a small problem appeared since we only had the Top10 strain at our disposal. But the New England Biolabs sponsorship arrived just in time with the DH5α strain, which allowed us to properly undertake all the protocols.</p></div> | |
− | </div> | + | <div class="a"><p align="justify">Due to the fact that we didn't have a microplate reader, taking part in the InterLab Study was the perfect opportunity to learn how to use one. The only drawback we had was getting the equipment to take the measurements but, in the end, the Research Center for Advanced Materials (CIMAV) lent us their laboratories. We are very proud because this year was the first time Tec-Chihuahua successfully participated in the InterLab Study.</p></div> |
+ | </div> | ||
+ | |||
<div class="a"> | <div class="a"> | ||
<p align="justify">The goal of the iGEM InterLab Study is to identify and correct the sources of systematic variability in synthetic biology measurements. In order to address this challenge, the iGEM committee provides researchers with detailed protocols and requires data analysis on the measurement of GFP. This year, the million dollar question is: <font color="#dd7a23"> Can we reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of OD? </font></p> | <p align="justify">The goal of the iGEM InterLab Study is to identify and correct the sources of systematic variability in synthetic biology measurements. In order to address this challenge, the iGEM committee provides researchers with detailed protocols and requires data analysis on the measurement of GFP. This year, the million dollar question is: <font color="#dd7a23"> Can we reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of OD? </font></p> | ||
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<p align="justify">For more information, check out <a href="https://2018.igem.org/Measurement/InterLab">iGEM's official InterLab Study webpage</a>. Below are the protocols we used and the data that we collected for the InterLab Study. </p> | <p align="justify">For more information, check out <a href="https://2018.igem.org/Measurement/InterLab">iGEM's official InterLab Study webpage</a>. Below are the protocols we used and the data that we collected for the InterLab Study. </p> | ||
− | < | + | |
+ | <h2 align="center">Calibration Measurements</h2> | ||
+ | <h3 align="center">OD<sub>600</sub> Reference Point</h3> | ||
+ | <div class="a"> | ||
+ | <p align="justify">The first calibration was to obtain a conversion factor to transform the absorbance data from the plate reader into a comparable OD<sub>600</sub> measurement, using LUDOX CL-X as a single point reference.</p> | ||
+ | </div> | ||
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<a data-toggle="collapse" data-parent="#accordion" href="#collapse1"><center> | <a data-toggle="collapse" data-parent="#accordion" href="#collapse1"><center> | ||
− | <div class="col-md-12"> | + | <div class="col-md-12">Calibration 1: OD600 Reference Point - LUDOX Protocol <div class="noDown" style="position: absolute; top: 30px; right: 30px;">▼</div></div></center></a> |
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+ | </div> | ||
+ | <br> | ||
<h3 align="center">Particle Standard Curve</h3> | <h3 align="center">Particle Standard Curve</h3> | ||
+ | <div class="a"> | ||
+ | <p align="justify">In the second calibration was obtained a standard curve of particle concentration which was used to convert Abs 600 measurements to an estimated number of cells. It was measured a dilution of monodisperse silica microspheres.</p></div> | ||
+ | <div class="panel panel-default"> | ||
+ | <div class="panel-heading"> | ||
+ | <h4 class="panel-title"> | ||
+ | <a data-toggle="collapse" data-parent="#accordion" href="#collapse2"><center> | ||
+ | <div class="col-md-12">Particle Standard Curve - Microsphere Protocol<div class="noDown" style="position: absolute; top: 30px; right: 30px;">▼</div></center></a> | ||
+ | </h4> | ||
+ | </div> | ||
+ | <div id="collapse2" class="panel-collapse collapse"> | ||
+ | <div class="panel-body"> | ||
+ | <object data="https://static.igem.org/mediawiki/2018/a/a4/T--Tec-Chihuahua--Cal2.pdf" type="application/pdf" width="100%" height="700px" align="middle"> </object> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | <br> | ||
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− | <h6 align="center"; style="font-size=" | + | <h6 align="center"; style="font-size="100%">Particle Standard Curve</h6> |
<img src="https://static.igem.org/mediawiki/2018/6/65/T--Tec-Chihuahua--Calibracion_2.1.png"> | <img src="https://static.igem.org/mediawiki/2018/6/65/T--Tec-Chihuahua--Calibracion_2.1.png"> | ||
</div> | </div> | ||
<div class="col-md-6"> | <div class="col-md-6"> | ||
− | <h6 align="center"; style="font-size=" | + | <h6 align="center"; style="font-size="100%">Particle Standard Curve (log scale)</h6> |
<img src="https://static.igem.org/mediawiki/2018/c/c3/T--Tec-Chihuahua--Calibracion_2.2.png"> | <img src="https://static.igem.org/mediawiki/2018/c/c3/T--Tec-Chihuahua--Calibracion_2.2.png"> | ||
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+ | <p><b>Particles/OD<sub>600</sub></b></p> | ||
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+ | <h3 align="center">Fluorescence Standard Curve</h3> | ||
+ | <div class="a"> | ||
+ | <p align="justify">In the third calibration, we generated a standard curve of fluorescence for fluorescein concentration, which was used to convert the cell-based readings to an equivalent fluorescein concentration.</p></div> | ||
− | < | + | <div class="panel panel-default"> |
+ | <div class="panel-heading"> | ||
+ | <h4 class="panel-title"> | ||
+ | <a data-toggle="collapse" data-parent="#accordion" href="#collapse3"><center> | ||
+ | <div class="col-md-12">Calibration 3: Fluorescence Standard Curve - Fluorescein Protocol<div class="noDown" style="position: absolute; top: 30px; right: 30px;">▼</div></center></a> | ||
+ | </h4> | ||
+ | </div> | ||
+ | <div id="collapse3" class="panel-collapse collapse"> | ||
+ | <div class="panel-body"> | ||
+ | <object data="https://static.igem.org/mediawiki/2018/1/18/T--Tec-Chihuahua--Cal3.pdf" type="application/pdf" width="100%" height="700px" align="middle"> </object> | ||
+ | </div> | ||
+ | </div> | ||
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</div><br><br> | </div><br><br> | ||
+ | <h3 align="center"><b>Results Part I: Cell Measurement</b></h3> | ||
+ | <div class="a"> | ||
+ | <p align="justify">After the calibrations protocols were done, we continued with the cell measurements which were made under the same condition. The results that were obtained are shown below.</p></div> | ||
+ | |||
+ | <div class="panel panel-default"> | ||
+ | <div class="panel-heading"> | ||
+ | <h4 class="panel-title"> | ||
+ | <a data-toggle="collapse" data-parent="#accordion" href="#collapse4"><center> | ||
+ | <div class="col-md-12">Cell Measurement Protocol<div class="noDown" style="position: absolute; top: 30px; right: 30px;">▼</div></center></a> | ||
+ | </h4> | ||
+ | </div> | ||
+ | <div id="collapse4" class="panel-collapse collapse"> | ||
+ | <div class="panel-body"> | ||
+ | <object data="https://static.igem.org/mediawiki/2018/1/17/T--Tec-Chihuahua--Cell.pdf" type="application/pdf" width="100%" height="700px" align="middle"> </object> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
<h3 align="center">Fluorescence Raw Readings</h3> | <h3 align="center">Fluorescence Raw Readings</h3> | ||
+ | <h4 align="center"><b>Hour 0</b></h4> | ||
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<th>Neg. Control</th> | <th>Neg. Control</th> | ||
<th>Pos. Control</th> | <th>Pos. Control</th> | ||
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</div><br><br> | </div><br><br> | ||
</div> | </div> | ||
+ | |||
+ | <div class="col-md-6"> | ||
+ | <img src="https://static.igem.org/mediawiki/2018/d/d3/T--Tec-Chihuahua--FL0.png"> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | <h4 align="center"><b>Hour 6<b></h4> | ||
+ | <div class="row"> | ||
+ | <div class="col-md-6"> | ||
+ | <img src="https://static.igem.org/mediawiki/2018/c/c2/T--Tec-Chihuahua--FL6.png"> | ||
+ | </div> | ||
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<h3 align="center">Abs600 Raw Readings</h3> | <h3 align="center">Abs600 Raw Readings</h3> | ||
+ | <h4 align="center"><b>Hour 0</b></h4> | ||
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</div> | </div> | ||
+ | </div> | ||
+ | <div class="col-md-6"> | ||
+ | <img src="https://static.igem.org/mediawiki/2018/5/52/T--Tec-Chihuahua--Abs0.png"> | ||
+ | </div> | ||
+ | </div> | ||
+ | <br> | ||
+ | |||
+ | <h4 align="center"><b>Hour 6</b></h4> | ||
+ | <div class="row"> | ||
+ | <div class="col-md-6"> | ||
+ | <img src="https://static.igem.org/mediawiki/2018/5/56/T--Tec-Chihuahua--Abs6.png"> | ||
+ | </div> | ||
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− | </div | + | </div> |
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+ | <br><br> | ||
− | < | + | <h3 align="center"><b>Results Part II: CFU per 0.1 OD<sub>600</sub></b></h3></b></b> |
+ | |||
+ | <div class="panel panel-default"> | ||
+ | <div class="panel-heading"> | ||
+ | <h4 class="panel-title"> | ||
+ | <a data-toggle="collapse" data-parent="#accordion" href="#collapse5"><center> | ||
+ | <div class="col-md-12">Colony Forming Units per 0.1 OD<sub>600</sub> <i>E. coli</i> Cultures Protocols<div class="noDown" style="position: absolute; top: 30px; right: 30px;">▼</div></center></a> | ||
+ | </h4> | ||
+ | </div> | ||
+ | <div id="collapse5" class="panel-collapse collapse"> | ||
+ | <div class="panel-body"> | ||
+ | <object data="https://static.igem.org/mediawiki/2018/4/49/T--Tec-Chihuahua--CFU.pdf" type="application/pdf" width="100%" height="700px" align="middle"> </object> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | <h3 align="justify">Dilution OD600 Measurements</h3> | ||
+ | <div class="a"><p align="justify">Thereafter, the protocol of colony forming units per 0.1 OD600 was made. The overnight cultures were diluted, obtaining the following results.</p></div> | ||
+ | <div style="overflow-x:auto;"> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <td></td> | ||
+ | <td colspan="3"><b>Culture 1</b></td> | ||
+ | <td colspan="3"><b>Culture 2</b></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td></td> | ||
+ | <td>Replicate 1</td> | ||
+ | <td>Replicate 2</td> | ||
+ | <td>Replicate 3</td> | ||
+ | <td>Replicate 1</td> | ||
+ | <td>Replicate 2</td> | ||
+ | <td>Replicate 3</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><b>Positive Control</b> | ||
+ | <td style="background-color:#ffe599"><b>0.099</b></td> | ||
+ | <td style="background-color:#ffe599"><b>0.105</b></td> | ||
+ | <td style="background-color:#ffe599"><b>0.102</b></td> | ||
+ | <td style="background-color:#ffe599"><b>0.097</b></td> | ||
+ | <td style="background-color:#ffe599"><b>0.100</b></td> | ||
+ | <td style="background-color:#ffe599"><b>0.096</b></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><b>Negative Control</b></td> | ||
+ | <td style="background-color:#ffe599"><b>0.109</b></td> | ||
+ | <td style="background-color:#ffe599"><b>0.103</b></td> | ||
+ | <td style="background-color:#ffe599"><b>0.105</b></td> | ||
+ | <td style="background-color:#ffe599"><b>0.101</b></td> | ||
+ | <td style="background-color:#ffe599"><b>0.101</b></td> | ||
+ | <td style="background-color:#ffe599"><b>0.099</b></td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <br><br> | ||
<h3 align="justify">Colony Counting for Dilution Factors Plates</h3> | <h3 align="justify">Colony Counting for Dilution Factors Plates</h3> | ||
+ | <div class="a"><p align="justify">Following this, serial dilutions were made and the last three were spread on plate. The number of colonies after 19 hours of growth were:</p></div> | ||
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<p><b>TNTC</b>="<b>T</b>oo <b>N</b>umerous <b>T</b>o <b>C</b>ount"</p> | <p><b>TNTC</b>="<b>T</b>oo <b>N</b>umerous <b>T</b>o <b>C</b>ount"</p> | ||
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<h3 align="justify">CFU Calculations</h3> | <h3 align="justify">CFU Calculations</h3> | ||
+ | <div class="a"><p align="justify">Finally, colony forming units (CFU) per 1mL of an OD600 = 0.1 culture were calculated. The results are as follows:</p></div> | ||
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Latest revision as of 04:10, 14 October 2018
![](https://static.igem.org/mediawiki/2018/3/34/T--Tec-Chihuahua--interlabletter.png)
All of the 2018 iGEM teams were invited to participate in the Fifth International InterLaboratory Measurement Study in synthetic biology. Team Tec-Chihuahua is excited about working on an international and collaborative experiment!
The protocols were actually very simple to understand and we didn't have any problems following them. Without a doubt, it was an enlightening experience full of challenges that helped us improve laboratory work during the development of the project. When we started working on it, a small problem appeared since we only had the Top10 strain at our disposal. But the New England Biolabs sponsorship arrived just in time with the DH5α strain, which allowed us to properly undertake all the protocols.
Due to the fact that we didn't have a microplate reader, taking part in the InterLab Study was the perfect opportunity to learn how to use one. The only drawback we had was getting the equipment to take the measurements but, in the end, the Research Center for Advanced Materials (CIMAV) lent us their laboratories. We are very proud because this year was the first time Tec-Chihuahua successfully participated in the InterLab Study.
The goal of the iGEM InterLab Study is to identify and correct the sources of systematic variability in synthetic biology measurements. In order to address this challenge, the iGEM committee provides researchers with detailed protocols and requires data analysis on the measurement of GFP. This year, the million dollar question is: Can we reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of OD?
For more information, check out iGEM's official InterLab Study webpage. Below are the protocols we used and the data that we collected for the InterLab Study.
Calibration Measurements
OD600 Reference Point
The first calibration was to obtain a conversion factor to transform the absorbance data from the plate reader into a comparable OD600 measurement, using LUDOX CL-X as a single point reference.
LUDOX CL-X | H2O | |
---|---|---|
Replicate 1 | 0.054 | 0.04 |
Replicate 2 | 0.051 | 0.035 |
Replicate 3 | 0.051 | 0.035 |
Replicate 4 | 0.052 | 0.035 |
Arith. Mean | 0.052 | 0.036 |
Corrected Abs600 | 0.016 | |
Reference OD600 | 0.063 | |
OD600/Abs600 | 4 |
Particle Standard Curve
In the second calibration was obtained a standard curve of particle concentration which was used to convert Abs 600 measurements to an estimated number of cells. It was measured a dilution of monodisperse silica microspheres.
Number of Particles | 2.35E+08 | 1.18E+08 | 5.88E+07 | 2.94E+07 | 1.47E+07 | 7.35E+06 | 3.68E+06 | 1.84E+06 | 9.19E+05 | 4.60E+05 | 2.30E+05 | 0 |
---|---|---|---|---|---|---|---|---|---|---|---|---|
Replicate 1 | 0.601 | 0.236 | 0.171 | 0.105 | 0.071 | 0.053 | 0.045 | 0.039 | 0.038 | 0.037 | 0.036 | 0.035 |
Replicate 2 | 0.49 | 0.263 | 0.156 | 0.102 | 0.068 | 0.052 | 0.044 | 0.041 | 0.039 | 0.036 | 0.034 | 0.037 |
Replicate 3 | 0.519 | 0.269 | 0.191 | 0.098 | 0.07 | 0.051 | 0.043 | 0.04 | 0.037 | 0.039 | 0.037 | 0.034 |
Replicate 4 | 0.516 | 0.269 | 0.171 | 0.102 | 0.071 | 0.054 | 0.044 | 0.039 | 0.039 | 0.036 | 0.035 | 0.034 |
Arith. Mean | 0.532 | 0.259 | 0.172 | 0.102 | 0.07 | 0.053 | 0.044 | 0.04 | 0.038 | 0.037 | 0.036 | 0.035 |
Arith. Std Dev. | 0.048 | 0.016 | 0.014 | 0.003 | 0.001 | 0.001 | 0.001 | 0.001 | 0.001 | 0.001 | 0.001 | 0.001 |
Arith. Net Mean | 0.497 | 0.224 | 0.137 | 0.067 | 0.035 | 0.018 | 0.009 | 0.005 | 0.003 | 0.002 | 0.001 |
Particle Standard Curve
![](https://static.igem.org/mediawiki/2018/6/65/T--Tec-Chihuahua--Calibracion_2.1.png)
Particle Standard Curve (log scale)
![](https://static.igem.org/mediawiki/2018/c/c3/T--Tec-Chihuahua--Calibracion_2.2.png)
Particles/OD600
Number of Particles | 2.35E+08 | 1.18E+08 | 5.88E+07 | 2.94E+07 | 1.47E+07 | 7.35E+06 | 3.68E+06 | 1.84E+06 | 9.19E+05 | 4.60E+05 | 2.30E+05 |
---|---|---|---|---|---|---|---|---|---|---|---|
Mean Particles/Abs600 | 4.74E+08 | 5.25E+08 | 4.29E+08 | 4.41E+08 | 4.20E+08 | 4.20E+08 | 4.08E+08 | 3.87E+08 | 2.83E+08 | 2.30E+08 | 4.60E+08 |
Mean of med-high levels | 4.47E+08 |
Fluorescence Standard Curve
In the third calibration, we generated a standard curve of fluorescence for fluorescein concentration, which was used to convert the cell-based readings to an equivalent fluorescein concentration.
Fluorescein uM | 10 | 5 | 2.5 | 1.25 | 0.625 | 0.313 | 0.156 | 0.078 | 0.039 | 0.0195 | 0.0098 | 0 |
---|---|---|---|---|---|---|---|---|---|---|---|---|
Replicate 1 | 1852 | 969.7 | 538.3 | 275.5 | 136.1 | 71.11 | 35.16 | 17.74 | 8.998 | 4.618 | 2.761 | 0.3263 |
Replicate 2 | 1929 | 956.8 | 523.7 | 284.7 | 132.6 | 66.21 | 36.66 | 17.95 | 8.892 | 4.856 | 2.553 | 0.34 |
Replicate 3 | 1922 | 977.1 | 516.5 | 281.1 | 150.3 | 75.65 | 40.22 | 19.54 | 10.57 | 5.299 | 2.931 | 0.3541 |
Replicate 4 | 1929 | 980.4 | 518.8 | 277.4 | 144.7 | 72.36 | 36.64 | 18.28 | 9.432 | 4.724 | 2.5 | 0.3667 |
Arith. Mean | 1.91E+03 | 9.71E+02 | 5.24E+02 | 2.80E+02 | 1.41E+02 | 7.13E+01 | 3.72E+01 | 1.84E+01 | 9.47E+00 | 4.87E+00 | 2.69E+00 | 3.47E-01 |
Arith. Std. Dev. | 3.75E+01 | 1.05E+01 | 9.79E+00 | 4.08E+00 | 8.06E+00 | 3.92E+00 | 2.15E+00 | 8.06E-01 | 7.68E-01 | 2.99E-01 | 1.98E-01 | 1.75E-02 |
Arith. Net Mean | 1.91E+03 | 9.71E+02 | 5.24E+02 | 2.79E+02 | 1.41E+02 | 7.10E+01 | 3.68E+01 | 1.80E+01 | 9.13E+00 | 4.53E+00 | 2.34E+00 |
Fluorescein Standard Curve
![](https://static.igem.org/mediawiki/2018/b/b5/T--Tec-Chihuahua--Calibracion_3.1.png)
Fluorescein Standard Curve (log scale)
![](https://static.igem.org/mediawiki/2018/0/05/T--Tec-Chihuahua--Calibracion_3.2.png)
Fluorescein uM | 10 | 5 | 2.5 | 1.25 | 0.63 | 0.31 | 0.16 | 0.08 | 0.04 | 0.02 | 0.01 |
---|---|---|---|---|---|---|---|---|---|---|---|
uM Fluorescein/a.u. | 5.24E-03 | 5.15E-03 | 4.77E-03 | 4.48E-03 | 4.45E-03 | 4.40E-03 | 4.24E-03 | 4.33E-03 | 4.28E-03 | 4.31E-03 | 4.17E-03 |
Mean uM Fluorescein/a.u. | 4.65E-03 | ||||||||||
MEFL/a.u. | 2.80E+10 |
Results Part I: Cell Measurement
After the calibrations protocols were done, we continued with the cell measurements which were made under the same condition. The results that were obtained are shown below.
Fluorescence Raw Readings
Hour 0
Neg. Control | Pos. Control | Device 1 | Device 2 | Device 3 | Device 4 | Device 5 | Device 6 | LB + Chlor (blank) | |
---|---|---|---|---|---|---|---|---|---|
Colony 1, Replicate 1 | 5.482 | 8.456 | 18.54 | 9.417 | 5.513 | 18.04 | 26.03 | 6.729 | 4.351 |
Colony 1, Replicate 2 | 4.915 | 8.344 | 18.3 | 9.125 | 5.328 | 17.97 | 25.61 | 6.249 | 4.244 |
Colony 1, Replicate 3 | 5.244 | 8.183 | 18.5 | 9.04v | 5.163 | 18.2v | 25.89 | 6.73 | 4.447 |
Colony 1, Replicate 4 | 5.426 | 8.369 | 17.98 | 8.748 | 5.434 | 17.18 | 25.73 | 6.637 | 4.321 |
Colony 2, Replicate 1 | 5.348 | 7.593 | 20.28 | 8.4 | 5.534 | 17.25 | 22.39 | 5.868 | 4.355 |
Colony 2, Replicate 2 | 5.211 | 7.304 | 18.93 | 8.121 | 5.333 | 16.32 | 21.94 | 5.796 | 4.402 |
Colony 2, Replicate 3 | 5.308 | 7.355 | 19.89 | 8.181 | 5.408 | 16.89 | 21.81 | 5.982 | 4.399 |
Colony 2, Replicate 4 | 5.376 | 7.487 | 19.38 | 8.325 | 5.272 | 16.78 | 21.2 | 5.992 | 4.458 |
![](https://static.igem.org/mediawiki/2018/d/d3/T--Tec-Chihuahua--FL0.png)
Hour 6
![](https://static.igem.org/mediawiki/2018/c/c2/T--Tec-Chihuahua--FL6.png)
Neg. Control | Pos. Control | Device 1 | Device 2 | Device 3 | Device 4 | Device 5 | Device 6 | LB + Chlor (blank) | |
---|---|---|---|---|---|---|---|---|---|
Colony 1, Replicate 1 | 5.216 | 11.56 | 45.15 | 8.42 | 5.249 | 37.7 | 44.03 | 7.702 | 2.608 |
Colony 1, Replicate 2 | 5.1 | 12.65 | 45.74 | 8.949 | 5.008 | 38.49 | 47.11 | 7.273 | 3.704 |
Colony 1, Replicate 3 | 5.051 | 12.33 | 45.71 | 14.25 | 5.063 | 37.05 | 46.05 | 7.627 | 3.382 |
Colony 1, Replicate 4 | 4.967 | 12.71 | 45.11 | 14.56 | 5.018 | 39.27 | 45.37 | 7.444 | 4.121 |
Colony 2, Replicate 1 | 4.917 | 12 | 46.24 | 11.25 | 5.11 | 40.01 | 41.62 | 6.062 | 3.163 |
Colony 2, Replicate 2 | 4.896 | 12.41 | 47.24 | 10.74 | 5.003 | 38.93 | 42.12 | 6.935 | 3.178 |
Colony 2, Replicate 3 | 4.973 | 12.03 | 47.16 | 10.94 | 5.224 | 40.58 | 40.07 | 6.718 | 3.464 |
Colony 2, Replicate 4 | 4.867 | 12.54 | 46.62 | 10.87 | 5.001 | 40.69 | 38.35 | 6.513 | 3.5 |
Abs600 Raw Readings
Hour 0
Neg. Control | Pos. Control | Device 1 | Device 2 | Device 3 | Device 4 | Device 5 | Device 6 | LB + Chlor (blank) | |
---|---|---|---|---|---|---|---|---|---|
Colony 1, Replicate 1 | 0.074 | 0.076 | 0.07 | 0.077 | 0.08 | 0.085 | 0.073 | 0.072 | 0.039 |
Colony 1, Replicate 2 | 0.073 | 0.079 | 0.07 | 0.072 | 0.073 | 0.075 | 0.073 | 0.074 | 0.041 |
Colony 1, Replicate 3 | 0.079 | 0.078 | 0.067 | 0.076 | 0.074 | 0.075 | 0.069 | 0.078 | 0.04 |
Colony 1, Replicate 4 | 0.077 | 0.082 | 0.073 | 0.077 | 0.079 | 0.072 | 0.071 | 0.077 | 0.04 |
Colony 2, Replicate 1 | 0.079 | 0.079 | 0.077 | 0.073 | 0.074 | 0.076 | 0.072 | 0.076 | 0.04 |
Colony 2, Replicate 2 | 0.079 | 0.075 | 0.072 | 0.07 | 0.071 | 0.069 | 0.07 | 0.071 | 0.04 |
Colony 2, Replicate 3 | 0.075 | 0.076 | 0.075 | 0.071 | 0.081 | 0.072 | 0.068 | 0.073 | 0.04 |
Colony 2, Replicate 4 | 0.074 | 0.076 | 0.073 | 0.067 | 0.073 | 0.076 | 0.093 | 0.073 | 0.039 |
![](https://static.igem.org/mediawiki/2018/5/52/T--Tec-Chihuahua--Abs0.png)
Hour 6
![](https://static.igem.org/mediawiki/2018/5/56/T--Tec-Chihuahua--Abs6.png)
Neg. Control | Pos. Control | Device 1 | Device 2 | Device 3 | Device 4 | Device 5 | Device 6 | LB + Chlor (blank) | |
---|---|---|---|---|---|---|---|---|---|
Colony 1, Replicate 1 | 0.176 | 0.155 | 0.187 | 0.113 | 0.177 | 0.16 | 0.122 | 0.152 | 0.037 |
Colony 1, Replicate 2 | 0.168 | 0.164 | 0.174 | 0.122 | 0.18 | 0.174 | 0.134 | 0.155 | 0.038 |
Colony 1, Replicate 3 | 0.169 | 0.154 | 0.166 | 0.174 | 0.182 | 0.161 | 0.129 | 0.154 | 0.037 |
Colony 1, Replicate 4 | 0.169 | 0.147 | 0.171 | 0.171 | 0.178 | 0.166 | 0.122 | 0.154 | 0.038 |
Colony 2, Replicate 1 | 0.172 | 0.175 | 0.173 | 0.163 | 0.18 | 0.185 | 0.129 | 0.136 | 0.038 |
Colony 2, Replicate 2 | 0.173 | 0.163 | 0.184 | 0.153 | 0.171 | 0.195 | 0.121 | 0.155 | 0.038 |
Colony 2, Replicate 3 | 0.178 | 0.165 | 0.178 | 0.166 | 0.185 | 0.208 | 0.132 | 0.148 | 0.038 |
Colony 2, Replicate 4 | 0.159 | 0.159 | 0.184 | 0.155 | 0.174 | 0.172 | 0.117 | 0.142 | 0.037 |
Results Part II: CFU per 0.1 OD600
Dilution OD600 Measurements
Thereafter, the protocol of colony forming units per 0.1 OD600 was made. The overnight cultures were diluted, obtaining the following results.
Culture 1 | Culture 2 | |||||
Replicate 1 | Replicate 2 | Replicate 3 | Replicate 1 | Replicate 2 | Replicate 3 | |
Positive Control | 0.099 | 0.105 | 0.102 | 0.097 | 0.100 | 0.096 |
Negative Control | 0.109 | 0.103 | 0.105 | 0.101 | 0.101 | 0.099 |
Colony Counting for Dilution Factors Plates
Following this, serial dilutions were made and the last three were spread on plate. The number of colonies after 19 hours of growth were:
Culture 1 | Culture 2 | ||||||
Replicate 1 | Replicate 2 | Replicate 3 | Replicate 1 | Replicate 2 | Replicate 3 | ||
Positive Control | 8.00E+04 | TNTC | TNTC | TNTC | TNTC | TNTC | TNTC |
8.00E+05 | 273 | TNTC | TNTC | 69 | 84 | 99 | |
8.00E+04 | 17 | TNTC | 41 | 9 | 35 | 20 | |
Negative Control | 8.00E+04 | TNTC | TNTC | TNTC | TNTC | TNTC | TNTC |
8.00E+05 | TNTC | TNTC | TNTC | TNTC | 283 | 300 | |
8.00E+04 | TNTC | 59 | 240 | 72 | 33 | 27 |
TNTC="Too Numerous To Count"
CFU Calculations
Finally, colony forming units (CFU) per 1mL of an OD600 = 0.1 culture were calculated. The results are as follows:
Culture 1 | Culture 2 | |||||
Replicate 1 | Replicate 2 | Replicate 3 | Replicate 1 | Replicate 2 | Replicate 3 | |
Positive Control | 1.36E+08 | TNTC | 3.28E+08 | 5.52E+07 | 6.72E+07 | 7.92E+07 |
Negative Control | TNTC | 4.72E+08 | 1.92E+09 | 5.76E+08 | 2.64E+08 | 2.16E+08 |
TNTC="Too Numerous To Count"