Difference between revisions of "Team:DTU-Denmark/InterLab"
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| − | + | <img src="https://static.igem.org/mediawiki/2018/d/dd/T--DTU-Denmark--topperinterlab.png" style="max-width: 100%; object-fit: cover; width:100%;" > | |
| + | <div class="headlinecontainer"><h1>InterLab</h1></div> | ||
| + | </div> | ||
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| − | <div class=" | + | <div class="igem_2018_team_content"> |
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| + | <!--<div class= "media media-body">--> | ||
<div class="interlabspace"> | <div class="interlabspace"> | ||
| − | <p> | + | <p style="text-align:justify"> |
To quote the official Interlab Measurement Study site, “reliable and repeatable measurement is a key component to all engineering disciplines”. <br><br> | To quote the official Interlab Measurement Study site, “reliable and repeatable measurement is a key component to all engineering disciplines”. <br><br> | ||
| − | As | + | As the first step in our wetlab efforts, we decided to embark on the fifth Interlab Measurement Study. In previous studies, it was shown that the variability between different lab measurements could be reduced by GFP fluorescence expression against a known concentration of a fluorescent molecule. However, the number of cells in the sample also holds a large impact on the variability.<br><br> |
| − | Therefore, this year’s | + | Therefore, this year’s InterLab study is about reducing the variability in fluorescence measurements between labs by normalizing to CFU or absolute cell count. To this, we firstly needed a plate reader that is capable of measuring both absorbance and fluorescence. We used the Biotek Synergy MX plate reader. Specifications of this model important to the protocol in use were:<br><br> |
</p> | </p> | ||
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| − | </ | + | <ul> |
| + | <li>It could measure both absorbance and fluorescence</li> | ||
| + | <li>It had path length correction</li> | ||
| + | <li>Temperature settings went from 4 to 65°C with a precision of ± 0.5°C at 37°C</li> | ||
| + | <li>Bandpass width was 530/20</li> | ||
| + | <li>Excitation was 485 nm</li> | ||
| + | <li>It used top optics</li> | ||
| + | </ul> | ||
| + | |||
| + | |||
</div> | </div> | ||
| + | |||
</div> | </div> | ||
| − | < | + | |
| + | <h2 style="color:#F8A05B;">Transformation of Competent Cells</h2> | ||
<hr> | <hr> | ||
<div class="interlabspace"> | <div class="interlabspace"> | ||
| − | <p> | + | <p style="text-align:justify"> |
| − | Luckily, we did not need to create our own competent cells as we already had high-efficiency dh5α cells at hand. As we had a sufficient amount of colonies on our plates, we picked two colonies from each plate and inoculated them in LB medium + chloramphenicol O/N at 37°C and 220 rpm. | + | Luckily, we did not need to create our own competent cells as we already had high-efficiency dh5α cells at hand. As we had a sufficient amount of colonies on our plates (fig. 1a illustrates our control), we picked two colonies from each plate and inoculated (see fig. 1b) them in LB medium + chloramphenicol O/N at 37°C and 220 rpm. |
</p> | </p> | ||
| − | <p style="text-align:center;"> <img src="https://static.igem.org/mediawiki/2018/e/ef/T--DTU-Denmark--interlab-comp-cells.jpg" style="max-width: | + | <p style="text-align:center;"> <img src="https://static.igem.org/mediawiki/2018/e/ef/T--DTU-Denmark--interlab-comp-cells.jpg" style="max-width: 75%;"> <figcaption><p style="text-align:center; font-size:14px;"><b>Fig. 1a: </b> LB-CAM control (100 py). <b>Fig. 1b: </b> Starting the overnight inoculation. </p></figcaption> |
</p> | </p> | ||
| + | </div> | ||
| − | + | ||
| − | < | + | |
| − | <hr> | + | |
| + | <h2 style="color:#F8A05B;">Calibration 1: OD600 Reference point - LUDOX Protocol</h2> | ||
| + | <hr class="hrstyle1"> | ||
<div class="interlabspace"> | <div class="interlabspace"> | ||
| − | <p> | + | <p style="text-align:justify"> |
| − | + | Afterward we measured the LUDOX CL-X (45% colloidal silica suspension) as a single point reference to gain a conversion factor to transform the absorbance to a OD600 measurement. Plate readers, in general, are volume dependent and this calibration was therefore necessary. We obtained the following data: | |
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<table class="tg"> | <table class="tg"> | ||
<tr> | <tr> | ||
| − | <th | + | <th></th> |
| − | <th | + | <th>LUDOX CL-X</th> |
| − | <th | + | <th>H2O</th> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td class="tg-mhe8 | + | <td class="tg-mhe8">Replicate 1<br></td> |
| − | <td | + | <td>0.051</td> |
| − | <td | + | <td>0.036</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td class="tg-mhe8 | + | <td class="tg-mhe8">Replicate 2</td> |
| − | <td | + | <td>0.052</td> |
| − | <td | + | <td>0.035</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td class="tg-mhe8"><span style="font-weight:300">Replicate 3</span></td> | <td class="tg-mhe8"><span style="font-weight:300">Replicate 3</span></td> | ||
| − | <td | + | <td>0.054</td> |
| − | <td | + | <td>0.037</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td class="tg-mhe8"><span style="font-weight:300">Replicate 4</span></td> | <td class="tg-mhe8"><span style="font-weight:300">Replicate 4</span></td> | ||
| − | <td | + | <td>0.052</td> |
| − | <td | + | <td>0.037</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td class="tg-mhe8"><span style="font-weight:300"> | + | <td class="tg-mhe8"><span style="font-weight:300">Arithmetic mean</span></td> |
| − | <td | + | <td>0.052</td> |
| − | <td | + | <td>0.036</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td class="tg-mhe8"><span style="font-weight:300">Corrected Abs600</span></td> | <td class="tg-mhe8"><span style="font-weight:300">Corrected Abs600</span></td> | ||
| − | <td | + | <td>0.016</td> |
| − | <td | + | <td></td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td class="tg-mhe8"><span style="font-weight:300">Reference OD600</span></td> | <td class="tg-mhe8"><span style="font-weight:300">Reference OD600</span></td> | ||
| − | <td | + | <td>0.063</td> |
| − | <td | + | <td></td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td class="tg-mhe8"><span style="font-weight:300">OD600/Abs600</span></td> | <td class="tg-mhe8"><span style="font-weight:300">OD600/Abs600</span></td> | ||
| − | <td | + | <td>3.938</td> |
| − | <td | + | <td></td> |
</tr> | </tr> | ||
</table> | </table> | ||
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</div> | </div> | ||
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| − | + | <h2 style="color:#F8A05B;">Calibration 2: Particle Standard Curve - Microsphere Protocol</h2> | |
| − | < | + | <hr class="hrstyle1"> |
| − | <hr> | + | |
<div class="interlabspace"> | <div class="interlabspace"> | ||
| − | <p> | + | <p style="text-align:justify"> |
| − | Next we prepared a dilution series of monodisperse silica microspheres to measure the Abs600 in our plate reader. Since there is a known amount of particles per volume and their size and optical characteristics are similar to those of cells, we could use the measurement to construct a standard curve. Our results were as | + | Next, we prepared a dilution series of monodisperse silica microspheres to measure the Abs600 in our plate reader. Since there is a known amount of particles per volume and their size and optical characteristics are similar to those of cells, we could use the measurement to construct a standard curve. Our results were as shown in fig. 2 and fig. 3: |
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| − | <p style="text-align:center;"> <img src="https://static.igem.org/mediawiki/2018/4/44/T--DTU-Denmark--interlab-graph1.png" style=" | + | <p style="text-align:center;"> <img src="https://static.igem.org/mediawiki/2018/4/44/T--DTU-Denmark--interlab-graph1.png" style="width: 80%;"><figcaption><p style="text-align:center; font-size:14px;"><b>Fig. 2: </b>Illustration of particle curve with the mean of med-high levels: 2.19E+08. </figcaption> |
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| − | <p style="text-align:center;"> <img src="https://static.igem.org/mediawiki/2018/1/1b/T--DTU-Denmark--interlab-graph2.png" style=" | + | <p style="text-align:center;"> <img src="https://static.igem.org/mediawiki/2018/1/1b/T--DTU-Denmark--interlab-graph2.png" style="width: 80%;"><figcaption><p style="text-align:center; font-size:14px;"><b>Fig. 3: </b>Illustration of particle curve as log with the mean of med-high levels: 2.19E+08 </figcaption> |
</p> | </p> | ||
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| − | < | + | <h2 style="color:#F8A05B;">Calibration 3: Fluorescence Standard Curve - Fluorescein Protocol</h2> |
| − | <hr> | + | <hr class="hrstyle1"> |
<div class="interlabspace"> | <div class="interlabspace"> | ||
| − | <p> | + | <p style="text-align:justify"> |
| − | In the next segment, we were asked to create a fluorescence standard curve using fluorescein. This was done in order to compare the fluorescence outputs from team to team. As the stability of the GFP protein, as well as the high cost for its purification, is a drawback, we used fluorescein with similar excitation and emission properties as GFP. By creating a dilution series, the following results were gained: | + | In the next segment, we were asked to create a fluorescence standard curve using fluorescein. This was done in order to compare the fluorescence outputs from team to team. As the stability of the GFP protein, as well as the high cost for its purification, is a drawback, we used fluorescein with similar excitation and emission properties as GFP. By creating a dilution series, the following results (see fig 4 and fig 5) were gained: |
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| − | <style | + | <p style="text-align:center;"> <img src="https://static.igem.org/mediawiki/2018/e/e8/T--DTU-Denmark--interlab-graph3.png" style="width: 80%;"><figcaption><p style="text-align:center; font-size:14px;"><b>Fig. 4: </b>Fluorescein standard curve with Mean uM fluorescein / a.u.: 2.33E-04. MEFL / a.u.: 1.40E+09. </figcaption> |
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| − | Mean uM fluorescein / a.u.: 2 | + | |
| − | MEFL / a.u.: 1 | + | |
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| − | </ | + | |
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| − | <p style="text-align:center;"> <img src="https://static.igem.org/mediawiki/2018/6/60/T--DTU-Denmark--interlab-graph4.png" style=" | + | <p style="text-align:center;"> <img src="https://static.igem.org/mediawiki/2018/6/60/T--DTU-Denmark--interlab-graph4.png" style="width: 80%;"><figcaption><p style="text-align:center; font-size:14px;"><b>Fig. 5: </b>Fluorescein standard curve as log with Mean uM fluorescein / a.u.: 2.33E-04. MEFL / a.u.: 1.40E+09 </figcaption> |
</p> | </p> | ||
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| − | < | + | <h2 style="color:#F8A05B;">Cell Measurement of Transformed Cells: OD600 and Fluorescence</h2> |
| − | <hr> | + | <hr class="hrstyle1"> |
<div class="interlabspace"> | <div class="interlabspace"> | ||
| − | <p> | + | <p style="text-align:justify"> |
After performing the calibrations with a pleasing result, we measured OD600 and fluorescence of the transformed cells at 0 and 6 hours. Measurements of given times were created and calculations with the values we obtained from the standard curves and reference points gave us the following data: | After performing the calibrations with a pleasing result, we measured OD600 and fluorescence of the transformed cells at 0 and 6 hours. Measurements of given times were created and calculations with the values we obtained from the standard curves and reference points gave us the following data: | ||
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</p> | </p> | ||
<h4>Flourescence Raw Readings</h4> | <h4>Flourescence Raw Readings</h4> | ||
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<table class="tg"> | <table class="tg"> | ||
<tr> | <tr> | ||
| − | <th | + | <th>Hour 0:</th> |
| − | <th | + | <th>Neg. Control</th> |
| − | <th | + | <th>Pos. Control</th> |
| − | <th | + | <th>Device 1</th> |
| − | <th | + | <th>Device 2</th> |
| − | <th | + | <th>Device 3</th> |
| − | <th | + | <th>Device 4</th> |
| − | <th | + | <th>Device 5</th> |
| − | <th | + | <th>Device 6</th> |
| − | <th | + | <th>LB + Chlor (blank)</th> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td class="tg-mhe8">Colony 1, Replicate 1</td> | <td class="tg-mhe8">Colony 1, Replicate 1</td> | ||
| − | <td | + | <td >47</td> |
| − | <td | + | <td>40</td> |
| − | <td | + | <td >141</td> |
| − | <td | + | <td>74</td> |
| − | <td | + | <td>13</td> |
| − | <td | + | <td>140</td> |
| − | <td | + | <td>102</td> |
| − | <td | + | <td>35</td> |
| − | <td | + | <td>15</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td class="tg-mhe8">Colony 1, Replicate 2</td> | <td class="tg-mhe8">Colony 1, Replicate 2</td> | ||
| − | <td | + | <td>0</td> |
| − | <td | + | <td>35</td> |
| − | <td | + | <td>161</td> |
| − | <td | + | <td>76</td> |
| − | <td | + | <td>25</td> |
| − | <td | + | <td>140</td> |
| − | <td | + | <td>78</td> |
| − | <td | + | <td>37</td> |
| − | <td | + | <td>38</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td class="tg-mhe8">Colony 1, Replicate 3</td> | <td class="tg-mhe8">Colony 1, Replicate 3</td> | ||
| − | <td | + | <td>3</td> |
| − | <td | + | <td>69</td> |
| − | <td | + | <td>84</td> |
| − | <td | + | <td>85</td> |
| − | <td | + | <td>26</td> |
| − | <td | + | <td>115</td> |
| − | <td | + | <td>71</td> |
| − | <td | + | <td>46</td> |
| − | <td | + | <td>19</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td class="tg-mhe8">Colony 1, Replicate 4</td> | <td class="tg-mhe8">Colony 1, Replicate 4</td> | ||
| − | <td | + | <td>3</td> |
| − | <td | + | <td>26</td> |
| − | <td | + | <td>87</td> |
| − | <td | + | <td>65</td> |
| − | <td | + | <td>25</td> |
| − | <td | + | <td>114</td> |
| − | <td | + | <td>68</td> |
| − | <td | + | <td>54</td> |
| − | <td | + | <td>45</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td class="tg-mhe8">Colony 2, Replicate 1</td> | <td class="tg-mhe8">Colony 2, Replicate 1</td> | ||
| − | <td | + | <td>24</td> |
| − | <td | + | <td>32</td> |
| − | <td | + | <td>139</td> |
| − | <td | + | <td>131</td> |
| − | <td | + | <td>40</td> |
| − | <td | + | <td>78</td> |
| − | <td | + | <td>114</td> |
| − | <td | + | <td>46</td> |
| − | <td | + | <td>15</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td class="tg-mhe8">Colony 2, Replicate 2</td> | <td class="tg-mhe8">Colony 2, Replicate 2</td> | ||
| − | <td | + | <td>43</td> |
| − | <td | + | <td>53</td> |
| − | <td | + | <td>145</td> |
| − | <td | + | <td>103</td> |
| − | <td | + | <td>24</td> |
| − | <td | + | <td>92</td> |
| − | <td | + | <td>92</td> |
| − | <td | + | <td>80</td> |
| − | <td | + | <td>26</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td class="tg-mhe8">Colony 2, Replicate 3</td> | <td class="tg-mhe8">Colony 2, Replicate 3</td> | ||
| − | <td | + | <td>16</td> |
| − | <td | + | <td>30</td> |
| − | <td | + | <td>117</td> |
| − | <td | + | <td>62</td> |
| − | <td | + | <td>23</td> |
| − | <td | + | <td>83</td> |
| − | <td | + | <td>85</td> |
| − | <td | + | <td>62</td> |
| − | <td | + | <td>5</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td class="tg-mhe8">Colony 2, Replicate 4</td> | <td class="tg-mhe8">Colony 2, Replicate 4</td> | ||
| − | <td | + | <td>2</td> |
| − | <td | + | <td>37</td> |
| − | <td | + | <td>105</td> |
| − | <td | + | <td>115</td> |
| − | <td | + | <td>16</td> |
| − | <td | + | <td>92</td> |
| − | <td | + | <td>72</td> |
| − | <td | + | <td>42</td> |
| − | <td | + | <td>27</td> |
</tr> | </tr> | ||
</table> | </table> | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
<table class="tg"> | <table class="tg"> | ||
<tr> | <tr> | ||
| − | <th | + | <th>Hour 6:</th> |
| − | <th | + | <th>Neg. Control</th> |
| − | <th | + | <th>Pos. Control</th> |
| − | <th | + | <th>Device 1</th> |
| − | <th | + | <th>Device 2</th> |
| − | <th | + | <th>Device 3</th> |
| − | <th | + | <th>Device 4</th> |
| − | <th | + | <th>Device 5</th> |
| − | <th | + | <th>Device 6</th> |
| − | <th | + | <th>LB + Chlor (blank)</th> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td class="tg-mhe8">Colony 1, Replicate 1</td> | <td class="tg-mhe8">Colony 1, Replicate 1</td> | ||
| − | <td | + | <td >9</td> |
| − | <td | + | <td>412</td> |
| − | <td | + | <td>196</td> |
| − | <td | + | <td>744</td> |
| − | <td | + | <td>32</td> |
| − | <td | + | <td>360</td> |
| − | <td | + | <td>120</td> |
| − | <td | + | <td>265</td> |
| − | <td | + | <td>39</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td class="tg-mhe8">Colony 1, Replicate 2</td> | <td class="tg-mhe8">Colony 1, Replicate 2</td> | ||
| − | <td | + | <td>28</td> |
| − | <td | + | <td>385</td> |
| − | <td | + | <td>235</td> |
| − | <td | + | <td>769</td> |
| − | <td | + | <td>29</td> |
| − | <td | + | <td>364</td> |
| − | <td | + | <td>197</td> |
| − | <td | + | <td>309</td> |
| − | <td | + | <td>24</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td class="tg-mhe8">Colony 1, Replicate 3</td> | <td class="tg-mhe8">Colony 1, Replicate 3</td> | ||
| − | <td | + | <td>30</td> |
| − | <td | + | <td>399</td> |
| − | <td | + | <td>264</td> |
| − | <td | + | <td >840</td> |
<td class="tg-0lax">10</td> | <td class="tg-0lax">10</td> | ||
<td class="tg-0lax">409</td> | <td class="tg-0lax">409</td> | ||
| Line 798: | Line 448: | ||
<h4>Abs600 Raw Readings</h4> | <h4>Abs600 Raw Readings</h4> | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
<table class="tg"> | <table class="tg"> | ||
<tr> | <tr> | ||
| − | <th | + | <th>Hour 0:</th> |
| − | <th | + | <th>Neg. Control</th> |
| − | <th | + | <th>Pos. Control</th> |
| − | <th | + | <th>Device 1</th> |
| − | <th | + | <th>Device 2</th> |
| − | <th | + | <th>Device 3</th> |
| − | <th | + | <th>Device 4</th> |
| − | <th | + | <th>Device 5</th> |
| − | <th | + | <th>Device 6</th> |
| − | <th | + | <th>LB + Chlor (blank)</th> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td class="tg-mhe8">Colony 1, Replicate 1</td> | <td class="tg-mhe8">Colony 1, Replicate 1</td> | ||
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.062</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.07</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.058</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.054</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.059</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.054</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.052</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.054</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.04</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td class="tg-mhe8">Colony 1, Replicate 2</td> | <td class="tg-mhe8">Colony 1, Replicate 2</td> | ||
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.058</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.061</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.063</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.058</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.063</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.058</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.05</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.054</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.041</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td class="tg-mhe8">Colony 1, Replicate 3</td> | <td class="tg-mhe8">Colony 1, Replicate 3</td> | ||
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.065</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.065</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.052</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.053</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.055</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.054</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.046</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.058</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.04</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td class="tg-mhe8">Colony 1, Replicate 4</td> | <td class="tg-mhe8">Colony 1, Replicate 4</td> | ||
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.056</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.057</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.053</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.055</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.056</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.079</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.059</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.053</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.04</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td class="tg-mhe8">Colony 2, Replicate 1</td> | <td class="tg-mhe8">Colony 2, Replicate 1</td> | ||
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.063</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.061</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.067</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.061</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.064</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.054</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.053</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.057</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.039</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td class="tg-mhe8">Colony 2, Replicate 2</td> | <td class="tg-mhe8">Colony 2, Replicate 2</td> | ||
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.063</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.066</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.058</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.061</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.062</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.056</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.049</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.052</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.039</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td class="tg-mhe8">Colony 2, Replicate 3</td> | <td class="tg-mhe8">Colony 2, Replicate 3</td> | ||
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.072</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.058</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.059</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.056</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.066</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.054</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.052</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.063</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.039</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td class="tg-mhe8">Colony 2, Replicate 4</td> | <td class="tg-mhe8">Colony 2, Replicate 4</td> | ||
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.066</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.06</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.052</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.059</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.062</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.051</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.048</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.06</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.041</td> |
</tr> | </tr> | ||
</table> | </table> | ||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
<table class="tg"> | <table class="tg"> | ||
<tr> | <tr> | ||
| − | <th | + | <th>Hour 6:</th> |
| − | <th | + | <th>Neg. Control</th> |
| − | <th | + | <th>Pos. Control</th> |
| − | <th | + | <th>Device 1</th> |
| − | <th | + | <th>Device 2</th> |
| − | <th | + | <th>Device 3</th> |
| − | <th | + | <th>Device 4</th> |
| − | <th | + | <th>Device 5</th> |
| − | <th | + | <th>Device 6</th> |
| − | <th | + | <th>LB + Chlor (blank)</th> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td class="tg-mhe8">Colony 1, Replicate 1</td> | <td class="tg-mhe8">Colony 1, Replicate 1</td> | ||
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.373</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.318</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.062</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.331</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.361</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.083</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.054</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.339</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.042</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td class="tg-mhe8">Colony 1, Replicate 2</td> | <td class="tg-mhe8">Colony 1, Replicate 2</td> | ||
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.393</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.345</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.069</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.353</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.357</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.083</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.058</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.35</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.04</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td class="tg-mhe8">Colony 1, Replicate 3</td> | <td class="tg-mhe8">Colony 1, Replicate 3</td> | ||
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.388</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.344</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.065</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.361</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.356</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.089</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.059</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.346</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.039</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td class="tg-mhe8">Colony 1, Replicate 4</td> | <td class="tg-mhe8">Colony 1, Replicate 4</td> | ||
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.377</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.334</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.066</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.338</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.359</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.091</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.058</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.368</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.043</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td class="tg-mhe8">Colony 2, Replicate 1</td> | <td class="tg-mhe8">Colony 2, Replicate 1</td> | ||
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.404</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.397</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.08</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.326</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.362</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.234</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.057</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.365</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.039</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td class="tg-mhe8">Colony 2, Replicate 2</td> | <td class="tg-mhe8">Colony 2, Replicate 2</td> | ||
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.37</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.331</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.082</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.295</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.339</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.219</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.056</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.324</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.039</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td class="tg-mhe8">Colony 2, Replicate 3</td> | <td class="tg-mhe8">Colony 2, Replicate 3</td> | ||
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.386</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.335</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.077</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.299</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.37</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.218</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.057</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.346</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.04</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td class="tg-mhe8">Colony 2, Replicate 4</td> | <td class="tg-mhe8">Colony 2, Replicate 4</td> | ||
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.402</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.379</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.079</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.33</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.364</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.23</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.056</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.376</td> |
| − | <td class="tg-0lax">0 | + | <td class="tg-0lax">0.04</td> |
</tr> | </tr> | ||
</table> | </table> | ||
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<tr> | <tr> | ||
| − | <th | + | <th colspan="2" >Unit Scaling Factors:</th> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td class="tg-mhe8">OD600 / Abs600</td> | <td class="tg-mhe8">OD600 / Abs600</td> | ||
| − | <td class="tg-0lax">3 | + | <td class="tg-0lax">3.94</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td class="tg-mhe8">uM Fluorescein / a.u.</td> | <td class="tg-mhe8">uM Fluorescein / a.u.</td> | ||
| − | <td class="tg-0lax">2 | + | <td class="tg-0lax">2.33E-04</td> |
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Latest revision as of 03:03, 18 October 2018
InterLab
To quote the official Interlab Measurement Study site, “reliable and repeatable measurement is a key component to all engineering disciplines”.
As the first step in our wetlab efforts, we decided to embark on the fifth Interlab Measurement Study. In previous studies, it was shown that the variability between different lab measurements could be reduced by GFP fluorescence expression against a known concentration of a fluorescent molecule. However, the number of cells in the sample also holds a large impact on the variability.
Therefore, this year’s InterLab study is about reducing the variability in fluorescence measurements between labs by normalizing to CFU or absolute cell count. To this, we firstly needed a plate reader that is capable of measuring both absorbance and fluorescence. We used the Biotek Synergy MX plate reader. Specifications of this model important to the protocol in use were:
- It could measure both absorbance and fluorescence
- It had path length correction
- Temperature settings went from 4 to 65°C with a precision of ± 0.5°C at 37°C
- Bandpass width was 530/20
- Excitation was 485 nm
- It used top optics
Transformation of Competent Cells
Luckily, we did not need to create our own competent cells as we already had high-efficiency dh5α cells at hand. As we had a sufficient amount of colonies on our plates (fig. 1a illustrates our control), we picked two colonies from each plate and inoculated (see fig. 1b) them in LB medium + chloramphenicol O/N at 37°C and 220 rpm.
Fig. 1a: LB-CAM control (100 py). Fig. 1b: Starting the overnight inoculation. 
Calibration 1: OD600 Reference point - LUDOX Protocol
Afterward we measured the LUDOX CL-X (45% colloidal silica suspension) as a single point reference to gain a conversion factor to transform the absorbance to a OD600 measurement. Plate readers, in general, are volume dependent and this calibration was therefore necessary. We obtained the following data:
| LUDOX CL-X | H2O | |
|---|---|---|
| Replicate 1 |
0.051 | 0.036 |
| Replicate 2 | 0.052 | 0.035 |
| Replicate 3 | 0.054 | 0.037 |
| Replicate 4 | 0.052 | 0.037 |
| Arithmetic mean | 0.052 | 0.036 |
| Corrected Abs600 | 0.016 | |
| Reference OD600 | 0.063 | |
| OD600/Abs600 | 3.938 |
Calibration 2: Particle Standard Curve - Microsphere Protocol
Next, we prepared a dilution series of monodisperse silica microspheres to measure the Abs600 in our plate reader. Since there is a known amount of particles per volume and their size and optical characteristics are similar to those of cells, we could use the measurement to construct a standard curve. Our results were as shown in fig. 2 and fig. 3:
Fig. 2: Illustration of particle curve with the mean of med-high levels: 2.19E+08. 
Fig. 3: Illustration of particle curve as log with the mean of med-high levels: 2.19E+08 
Calibration 3: Fluorescence Standard Curve - Fluorescein Protocol
In the next segment, we were asked to create a fluorescence standard curve using fluorescein. This was done in order to compare the fluorescence outputs from team to team. As the stability of the GFP protein, as well as the high cost for its purification, is a drawback, we used fluorescein with similar excitation and emission properties as GFP. By creating a dilution series, the following results (see fig 4 and fig 5) were gained:
Fig. 4: Fluorescein standard curve with Mean uM fluorescein / a.u.: 2.33E-04. MEFL / a.u.: 1.40E+09. 
Fig. 5: Fluorescein standard curve as log with Mean uM fluorescein / a.u.: 2.33E-04. MEFL / a.u.: 1.40E+09 
Cell Measurement of Transformed Cells: OD600 and Fluorescence
After performing the calibrations with a pleasing result, we measured OD600 and fluorescence of the transformed cells at 0 and 6 hours. Measurements of given times were created and calculations with the values we obtained from the standard curves and reference points gave us the following data:
Flourescence 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 | 47 | 40 | 141 | 74 | 13 | 140 | 102 | 35 | 15 |
| Colony 1, Replicate 2 | 0 | 35 | 161 | 76 | 25 | 140 | 78 | 37 | 38 |
| Colony 1, Replicate 3 | 3 | 69 | 84 | 85 | 26 | 115 | 71 | 46 | 19 |
| Colony 1, Replicate 4 | 3 | 26 | 87 | 65 | 25 | 114 | 68 | 54 | 45 |
| Colony 2, Replicate 1 | 24 | 32 | 139 | 131 | 40 | 78 | 114 | 46 | 15 |
| Colony 2, Replicate 2 | 43 | 53 | 145 | 103 | 24 | 92 | 92 | 80 | 26 |
| Colony 2, Replicate 3 | 16 | 30 | 117 | 62 | 23 | 83 | 85 | 62 | 5 |
| Colony 2, Replicate 4 | 2 | 37 | 105 | 115 | 16 | 92 | 72 | 42 | 27 |
| Hour 6: | Neg. Control | Pos. Control | Device 1 | Device 2 | Device 3 | Device 4 | Device 5 | Device 6 | LB + Chlor (blank) |
|---|---|---|---|---|---|---|---|---|---|
| Colony 1, Replicate 1 | 9 | 412 | 196 | 744 | 32 | 360 | 120 | 265 | 39 |
| Colony 1, Replicate 2 | 28 | 385 | 235 | 769 | 29 | 364 | 197 | 309 | 24 |
| Colony 1, Replicate 3 | 30 | 399 | 264 | 840 | 10 | 409 | 170 | 339 | 29 |
| Colony 1, Replicate 4 | 0 | 423 | 276 | 803 | 21 | 419 | 150 | 317 | 17 |
| Colony 2, Replicate 1 | 15 | 500 | 304 | 754 | 43 | 971 | 159 | 342 | 20 |
| Colony 2, Replicate 2 | 37 | 469 | 319 | 778 | 20 | 905 | 162 | 316 | 9 |
| Colony 2, Replicate 3 | 36 | 522 | 325 | 846 | 39 | 931 | 144 | 333 | 11 |
| Colony 2, Replicate 4 | 38 | 535 | 286 | 864 | 14 | 968 | 167 | 388 | 29 |
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 | 0.062 | 0.07 | 0.058 | 0.054 | 0.059 | 0.054 | 0.052 | 0.054 | 0.04 |
| Colony 1, Replicate 2 | 0.058 | 0.061 | 0.063 | 0.058 | 0.063 | 0.058 | 0.05 | 0.054 | 0.041 |
| Colony 1, Replicate 3 | 0.065 | 0.065 | 0.052 | 0.053 | 0.055 | 0.054 | 0.046 | 0.058 | 0.04 |
| Colony 1, Replicate 4 | 0.056 | 0.057 | 0.053 | 0.055 | 0.056 | 0.079 | 0.059 | 0.053 | 0.04 |
| Colony 2, Replicate 1 | 0.063 | 0.061 | 0.067 | 0.061 | 0.064 | 0.054 | 0.053 | 0.057 | 0.039 |
| Colony 2, Replicate 2 | 0.063 | 0.066 | 0.058 | 0.061 | 0.062 | 0.056 | 0.049 | 0.052 | 0.039 |
| Colony 2, Replicate 3 | 0.072 | 0.058 | 0.059 | 0.056 | 0.066 | 0.054 | 0.052 | 0.063 | 0.039 |
| Colony 2, Replicate 4 | 0.066 | 0.06 | 0.052 | 0.059 | 0.062 | 0.051 | 0.048 | 0.06 | 0.041 |
| Hour 6: | Neg. Control | Pos. Control | Device 1 | Device 2 | Device 3 | Device 4 | Device 5 | Device 6 | LB + Chlor (blank) |
|---|---|---|---|---|---|---|---|---|---|
| Colony 1, Replicate 1 | 0.373 | 0.318 | 0.062 | 0.331 | 0.361 | 0.083 | 0.054 | 0.339 | 0.042 |
| Colony 1, Replicate 2 | 0.393 | 0.345 | 0.069 | 0.353 | 0.357 | 0.083 | 0.058 | 0.35 | 0.04 |
| Colony 1, Replicate 3 | 0.388 | 0.344 | 0.065 | 0.361 | 0.356 | 0.089 | 0.059 | 0.346 | 0.039 |
| Colony 1, Replicate 4 | 0.377 | 0.334 | 0.066 | 0.338 | 0.359 | 0.091 | 0.058 | 0.368 | 0.043 |
| Colony 2, Replicate 1 | 0.404 | 0.397 | 0.08 | 0.326 | 0.362 | 0.234 | 0.057 | 0.365 | 0.039 |
| Colony 2, Replicate 2 | 0.37 | 0.331 | 0.082 | 0.295 | 0.339 | 0.219 | 0.056 | 0.324 | 0.039 |
| Colony 2, Replicate 3 | 0.386 | 0.335 | 0.077 | 0.299 | 0.37 | 0.218 | 0.057 | 0.346 | 0.04 |
| Colony 2, Replicate 4 | 0.402 | 0.379 | 0.079 | 0.33 | 0.364 | 0.23 | 0.056 | 0.376 | 0.04 |
| Unit Scaling Factors: | |
|---|---|
| OD600 / Abs600 | 3.94 |
| uM Fluorescein / a.u. | 2.33E-04 |
By these data, we can conclude that the InterLab study was successful and the InterLab study protocol can be used for standardization.