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<h1 class="mdc-typography--display1">Introduction</h1>
<h2 class="mdc-typography--headline">
Background
<!-- <span class="mdc-typography--caption">Caption.</span> -->
</h2>
<p class="mdc-typography--body2">
Many teachers and iGEM Headquarters says that reliable and repeatable measurement is a key component to all engineering disciplines. But to be honest it's difficult for laboratories all around the world to measure something in the same standard, so iGEM developing a robust measurement procedure for green fluorescent protein (GFP) every years to lead the team all over world could measure it.
</p>
<p class="mdc-typography--body2">
It's the fourth year for iGEM to require teams which participate in finishing the interlab work. And GFP is a most widely used.
</p>
<p class="mdc-typography--body2">
"All of the 2017 iGEM teams are invited and encouraged to participate in the Fourth International InterLaboratory Measurement Study in synthetic biology. We're hoping this study will get you excited for iGEM and help prepare you for the summer!" says by iGEM Headquarters. Actually, for us the interlab work not only to make fun but the more important part is that it can lead different laboratory to normalize their measure methods and machines so that we can use data from other lab easily, which will benefit our experiment and project a lot.
</p>
<p class="mdc-typography--body2">
We all glad to participate in such significant international work with those friendly workmates all around world.
</p>
<h2 class="mdc-typography--headline">Materials and methods</h2>
<h3 class="mdc-typography--title">Materials</h3>
<ul class="page__article__list">
<li>Plasmid DNA (1ng in total, 100 pg/uL in 10uL of ddH20)
<ul>
<li>Positive control BBa_I20270</li>
<li>Negative control BBa_R0040</li>
<li>Test Device 1: J23101.BCD2.E0040.B0015</li>
<li>Test Device 2: J23106.BCD2.E0040.B0015</li>
<li>Test Device 3: J23117.BCD2.E0040.B0015</li>
<li>Test Device 4: J23101+I13504</li>
<li>Test Device 5: J23106+I13504</li>
<li>Test Device 6: J23117+I13504</li>
</ul>
</li>
<li>Strain Used
<ul>
<li>Escherichia coli DH5α</li>
</ul>
</li>
<li>Media Used
<ul>
<li>LB (Luria Bertani) media</li>
</ul>
</li>
<li>Reagent Used
<ul>
<li>1xPBS (phosphate buffered saline)</li>
<li>Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH)</li>
<li>FITC Standard: one tube with dried down FITC for creating a FITC standard</li>
<li>LUDOX: one tube with 30% colloidal silica suspended in 1mL of water</li>
</ul>
</li>
<li>Consumable Items
<ul>
<li>50 ml Falcon tube</li>
<li>1.5 ml eppendorf tubes for sample storage</li>
<li>Ice box with ice</li>
<li>Pipettes and tip</li>
<li>96 well plate</li>
</ul>
</li>
</ul>
<h3 class="mdc-typography--title">Machines</h3>
<ul class="page__article__list">
<li>
<b>Thermo scientific</b>
Varioskan Flash
</li>
<li>
<b>CRYSTAL</b>
Incubator Shaker
</li>
<li>
<b>Yiheng-China</b>
HZQ-F160A constant temperature incubator
</li>
<li>
<b>AIRTECH</b>
Vertical Flow Clean Bench
</li>
</ul>
<h3 class="mdc-typography--title">Methods</h3>
<ul class="page__article__list">
<li>Calibration
<ul>
<li>OD600 Reference poin</li>
<li>FITC fluorescence standard curve</li>
</ul>
</li>
<li>Cell measurement
<ul>
<li>Transformation</li>
<li>Measurements</li>
</ul>
</li>
</ul>
<h1 class="mdc-typography--display1">Data and Analysis</h1>
<h2 class="mdc-typography--headline">Normalization work</h2>
<figure>
<table>
<thead>
<tr>
<th></th>
<th>LUDOX-HS40</th>
<th>H20</th>
</tr>
</thead>
<tbody>
<tr>
<th>Replicate 1</th>
<td>0.0546438</td>
<td>0.47919</td>
</tr>
<tr>
<th>Replicate 2</th>
<td>0.0646164</td>
<td>0.039539</td>
</tr>
<tr>
<th>Replicate 3</th>
<td>0.0528094</td>
<td>0.062382</td>
</tr>
<tr>
<th>Replicate 4</th>
<td>0.0468351</td>
<td>0.054382</td>
</tr>
<tr>
<th>Arith.Mean</th>
<td class="td-highlight">0.05472618</td>
<td class="td-highlight">0.051055</td>
</tr>
<tr>
<th>Corrected Abs600</th>
<td class="td-highlight" style="color: red;">0.0036709</td>
<td></td>
</tr>
<tr>
<th>Reference OD600</th>
<td class="td-highlight">0.00425</td>
<td></td>
</tr>
<tr>
<th>OD600/Abs600</th>
<td class="td-highlight">11.5775423</td>
<td></td>
</tr>
</tbody>
</table>
<figcaption>Table 1. OD600 Reference Point.</figcaption>
</figure>
<figure>
<div id="container-1"></div>
<figcaption>Figure 1. FITC standard curve.</figcaption><span> There is an approximate linear trend for increased fluorescence as [FITC] increases.</span>>
</figure>
<figure>
<div id="container-2"></div>
<figcaption>Figure 2. FITC standard curve (log scale).</figcaption><span> After changing the curve into log scale, the linear trend becomes more remarkable and reliable.</span>
</figure>
<figure>
<table>
<thead>
<tr>
<th>Unit Scaling Factors:</th>
<th></th>
</tr>
</thead>
<tbody>
<tr>
<th>OD600/Abs600</th>
<td class="td-highlight">11.58</td>
</tr>
<tr>
<th>uM Fluorescein/a.u.</th>
<td class="td-highlight">0.003458</td>
</tr>
</tbody>
</table>
<figcaption>Table 4. Normalization of initial concentration of colony.</figcaption><span> In order to unify the initial concentration before , we use Varioskan Flash to measure the OD600 after incubating overnight.</span>
</figure>
<h2 class="mdc-typography--headline">Cell Measurement</h2>
<p class="mdc-typography--body2">Because of the large account of data, we only use an average data in this form. (Average of 4 replicates of one colony)</p>
<figure>
<table>
<thead>
<tr>
<th>Abs600</th>
<th>Replicate</th>
<th>0h</th>
<th>2h</th>
<th>4h</th>
<th>6h</th>
</tr>
</thead>
<tbody>
<tr>
<th rowspan="2">Negative control</th>
<th>Colony 1</th>
<td>0.0666</td>
<td>0.1893</td>
<td>0.2708</td>
<td>0.4942</td>
</tr>
<tr>
<th>Colony 2</th>
<td>0.0615</td>
<td>0.1929</td>
<td>0.3000</td>
<td>0.5681</td>
</tr>
<tr>
<th rowspan="2">Positive Control</th>
<th>Colony 1</th>
<td>0.0601</td>
<td>0.1638</td>
<td>0.2949</td>
<td>0.5425</td>
</tr>
<tr>
<th>Colony 2</th>
<td>0.0613</td>
<td>0.1839</td>
<td>0.2982</td>
<td>0.5815</td>
</tr>
<tr>
<th rowspan="2">Test Device1</th>
<th>Colony 1</th>
<td>0.0647</td>
<td>0.0649</td>
<td>0.0757</td>
<td>0.0816</td>
</tr>
<tr>
<th>Colony 2</th>
<td>0.0560</td>
<td>0.0612</td>
<td>0.0627</td>
<td>0.0732</td>
</tr>
<tr>
<th rowspan="2">Test Device2</th>
<th>Colony 1</th>
<td>0.0616</td>
<td>0.1685</td>
<td>0.2480</td>
<td>0.4316</td>
</tr>
<tr>
<th>Colony 2</th>
<td>0.0607</td>
<td>0.1928</td>
<td>0.2977</td>
<td>0.4638</td>
</tr>
<tr>
<th rowspan="2">Test Device3</th>
<th>Colony 1</th>
<td>0.0463</td>
<td>0.1566</td>
<td>0.3120</td>
<td>0.4887</td>
</tr>
<tr>
<th>Colony 2</th>
<td>0.0521</td>
<td>0.1291</td>
<td>0.3464</td>
<td>0.6376</td>
</tr>
<tr>
<th rowspan="2">Test Device4</th>
<th>Colony 1</th>
<td>0.0571</td>
<td>0.1329</td>
<td>0.2899</td>
<td>0.4263</td>
</tr>
<tr>
<th>Colony 2</th>
<td>0.0551</td>
<td>0.1259</td>
<td>0.2656</td>
<td>0.5216</td>
</tr>
<tr>
<th rowspan="2">Test Device5</th>
<th>Colony 1</th>
<td>0.0528</td>
<td>0.1776</td>
<td>0.3140</td>
<td>0.5123</td>
</tr>
<tr>
<th>Colony 2</th>
<td>0.0655</td>
<td>0.2286</td>
<td>0.3704</td>
<td>0.5788</td>
</tr>
<tr>
<th rowspan="2">Test Device6</th>
<th>Colony 1</th>
<td>0.0575</td>
<td>0.1316</td>
<td>0.2915</td>
<td>0.5249</td>
</tr>
<tr>
<th>Colony 2</th>
<td>0.0536</td>
<td>0.1530</td>
<td>0.3032</td>
<td>0.5852</td>
</tr>
</tbody>
</table>
<figcaption>Tablet5. Raw Abs600 measurement.</figcaption>
</figure>
<figure>
<table>
<thead>
<tr>
<th>Abs600</th>
<th>Replicate</th>
<th>0h</th>
<th>2h</th>
<th>4h</th>
<th>6h</th>
</tr>
</thead>
<tbody>
<tr>
<th rowspan="2">Negative control</th>
<th>Colony 1</th>
<td>0.0256</td>
<td>0.1483</td>
<td>0.2298</td>
<td>0.4942</td>
</tr>
<tr>
<th>Colony 2</th>
<td>0.0205</td>
<td>0.1518</td>
<td>0.2590</td>
<td>0.5271</td>
</tr>
<tr>
<th rowspan="2">Positive Control</th>
<th>Colony 1</th>
<td>0.0191</td>
<td>0.1227</td>
<td>0.2539</td>
<td>0.5014</td>
</tr>
<tr>
<th>Colony 2</th>
<td>0.0203</td>
<td>0.1429</td>
<td>0.2571</td>
<td>0.5404</td>
</tr>
<tr>
<th rowspan="2">Test Device1</th>
<th>Colony 1</th>
<td>0.0237</td>
<td>0.0239</td>
<td>0.0347</td>
<td>0.0405</td>
</tr>
<tr>
<th>Colony 2</th>
<td>0.0149</td>
<td>0.0202</td>
<td>0.0217</td>
<td>0.0321</td>
</tr>
<tr>
<th rowspan="2">Test Device2</th>
<th>Colony 1</th>
<td>0.0206</td>
<td>0.1274</td>
<td>0.2069</td>
<td>0.3905</td>
</tr>
<tr>
<th>Colony 2</th>
<td>0.0197</td>
<td>0.1518</td>
<td>0.2567</td>
<td>0.4228</td>
</tr>
<tr>
<th rowspan="2">Test Device3</th>
<th>Colony 1</th>
<td>0.0053</td>
<td>0.1156</td>
<td>0.2710</td>
<td>0.4477</td>
</tr>
<tr>
<th>Colony 2</th>
<td>0.0110</td>
<td>0.0881</td>
<td>0.3053</td>
<td>0.5966</td>
</tr>
<tr>
<th rowspan="2">Test Device4</th>
<th>Colony 1</th>
<td>0.0161</td>
<td>0.0919</td>
<td>0.2489</td>
<td>0.3852</td>
</tr>
<tr>
<th>Colony 2</th>
<td>0.0141</td>
<td>0.0849</td>
<td>0.2246</td>
<td>0.4806</td>
</tr>
<tr>
<th rowspan="2">Test Device5</th>
<th>Colony 1</th>
<td>0.0118</td>
<td>0.1365</td>
<td>0.2730</td>
<td>0.4713</td>
</tr>
<tr>
<th>Colony 2</th>
<td>0.0244</td>
<td>0.1875</td>
<td>0.3294</td>
<td>0.5378</td>
</tr>
<tr>
<th rowspan="2">Test Device6</th>
<th>Colony 1</th>
<td>0.0165</td>
<td>0.0906</td>
<td>0.2505</td>
<td>0.4839</td>
</tr>
<tr>
<th>Colony 2</th>
<td>0.0126</td>
<td>0.1120</td>
<td>0.2622</td>
<td>0.5442</td>
</tr>
</tbody>
</table>
<figcaption>Table 6. Abs600 measurement after blank subtraction and correction.</figcaption>
</figure>
<figure>
<div id="container-3"></div>
<figcaption>Figure 3. Abs600 measurement after blank subtraction and correction.</figcaption><span> We can find that two colony of device 1 are very different with other colonies.</span>>
</figure>
<figure>
<table>
<thead>
<tr>
<th>Fluorescence</th>
<th>Replicate</th>
<th>0h</th>
<th>2h</th>
<th>4h</th>
<th>6h</th>
</tr>
</thead>
<tbody>
<tr>
<th rowspan="2">Negative control</th>
<th>Colony 1</th>
<td>8.4338</td>
<td>8.3597</td>
<td>8.3132</td>
<td>8.2557</td>
</tr>
<tr>
<th>Colony 2</th>
<td>8.1640</td>
<td>8.2490</td>
<td>8.2382</td>
<td>8.5850</td>
</tr>
<tr>
<th rowspan="2">Positive Control</th>
<th>Colony 1</th>
<td>12.904</td>
<td>52.145</td>
<td>75.724</td>
<td>128.86</td>
</tr>
<tr>
<th>Colony 2</th>
<td>12.139</td>
<td>51.145</td>
<td>70.542</td>
<td>135.21</td>
</tr>
<tr>
<th rowspan="2">Test Device1</th>
<th>Colony 1</th>
<td>40.412</td>
<td>51.952</td>
<td>65.562</td>
<td>76.598</td>
</tr>
<tr>
<th>Colony 2</th>
<td>28.957</td>
<td>41.592</td>
<td>45.161</td>
<td>54.124</td>
</tr>
<tr>
<th rowspan="2">Test Device2</th>
<th>Colony 1</th>
<td>14.583</td>
<td>55.274</td>
<td>93.031</td>
<td>184.99</td>
</tr>
<tr>
<th>Colony 2</th>
<td>12.624</td>
<td>51.857</td>
<td>88.926</td>
<td>174.29</td>
</tr>
<tr>
<th rowspan="2">Test Device3</th>
<th>Colony 1</th>
<td>8.3959</td>
<td>8.6644</td>
<td>9.1428</td>
<td>9.9313</td>
</tr>
<tr>
<th>Colony 2</th>
<td>7.8957</td>
<td>8.2509</td>
<td>9.4031</td>
<td>11.753</td>
</tr>
<tr>
<th rowspan="2">Test Device4</th>
<th>Colony 1</th>
<td>10.542</td>
<td>55.101</td>
<td>81.432</td>
<td>118.27</td>
</tr>
<tr>
<th>Colony 2</th>
<td>10.275</td>
<td>50.620</td>
<td>76.936</td>
<td>135.14</td>
</tr>
<tr>
<th rowspan="2">Test Device5</th>
<th>Colony 1</th>
<td>8.1853</td>
<td>15.857</td>
<td>18.422</td>
<td>23.463</td>
</tr>
<tr>
<th>Colony 2</th>
<td>8.3564</td>
<td>18.187</td>
<td>20.175</td>
<td>26.058</td>
</tr>
<tr>
<th rowspan="2">Test Device6</th>
<th>Colony 1</th>
<td>7.9647</td>
<td>8.1582</td>
<td>8.2918</td>
<td>8.8606</td>
</tr>
<tr>
<th>Colony 2</th>
<td>7.8015</td>
<td>8.0578</td>
<td>8.2781</td>
<td>8.7673</td>
</tr>
</tbody>
</table>
<figcaption>Table 7. Raw data of fluorescence measurement.</figcaption>
</figure>
<figure>
<table>
<thead>
<tr>
<th>Fluorescence</th>
<th>Replicate</th>
<th>0h</th>
<th>2h</th>
<th>4h</th>
<th>6h</th>
</tr>
</thead>
<tbody>
<tr>
<th rowspan="2">Negative control</th>
<th>Colony 1</th>
<td>0.5088</td>
<td>0.4347</td>
<td>0.3882</td>
<td>0.3307</td>
</tr>
<tr>
<th>Colony 2</th>
<td>0.2390</td>
<td>0.3240</td>
<td>0.3132</td>
<td>0.6600</td>
</tr>
<tr>
<th rowspan="2">Positive Control</th>
<th>Colony 1</th>
<td>4.9786</td>
<td>44.220</td>
<td>67.799</td>
<td>120.94</td>
</tr>
<tr>
<th>Colony 2</th>
<td>4.2144</td>
<td>43.220</td>
<td>62.617</td>
<td>127.29</td>
</tr>
<tr>
<th rowspan="2">Test Device1</th>
<th>Colony 1</th>
<td>32.487</td>
<td>44.027</td>
<td>57.637</td>
<td>68.673</td>
</tr>
<tr>
<th>Colony 2</th>
<td>21.032</td>
<td>33.667</td>
<td>37.236</td>
<td>46.199</td>
</tr>
<tr>
<th rowspan="2">Test Device2</th>
<th>Colony 1</th>
<td>6.6580</td>
<td>47.349</td>
<td>85.106</td>
<td>177.06</td>
</tr>
<tr>
<th>Colony 2</th>
<td>4.6994</td>
<td>43.932</td>
<td>81.001</td>
<td>166.37</td>
</tr>
<tr>
<th rowspan="2">Test Device3</th>
<th>Colony 1</th>
<td>0.4709</td>
<td>0.7394</td>
<td>1.2178</td>
<td>2.0063</td>
</tr>
<tr>
<th>Colony 2</th>
<td>-0.0293</td>
<td>0.3259</td>
<td>1.4781</td>
<td>3.8283</td>
</tr>
<tr>
<th rowspan="2">Test Device4</th>
<th>Colony 1</th>
<td>2.6174</td>
<td>47.176</td>
<td>73.507</td>
<td>110.34</td>
</tr>
<tr>
<th>Colony 2</th>
<td>2.3496</td>
<td>42.695</td>
<td>69.011</td>
<td>127.21</td>
</tr>
<tr>
<th rowspan="2">Test Device5</th>
<th>Colony 1</th>
<td>0.2603</td>
<td>7.9322</td>
<td>10.497</td>
<td>15.538</td>
</tr>
<tr>
<th>Colony 2</th>
<td>0.4314</td>
<td>10.262</td>
<td>12.250</td>
<td>18.133</td>
</tr>
<tr>
<th rowspan="2">Test Device6</th>
<th>Colony 1</th>
<td>0.0397</td>
<td>0.2332</td>
<td>0.3668</td>
<td>0.9356</td>
</tr>
<tr>
<th>Colony 2</th>
<td>-0.1235</td>
<td>0.1328</td>
<td>0.3531</td>
<td>0.8423</td>
</tr>
</tbody>
</table>
<figcaption>Table 8. Fluorescence measurement after blank subtraction and correction.</figcaption>
</figure>
<figure>
<div id="container-4"></div>
<figcaption>Figure 4. Fluorescence measurement after blank subtraction and correction.</figcaption>
</figure>
<figure>
<div id="container-5"></div>
<p>You can select area to zoom it.</p>
<figcaption>Figure 5. Average level of devices.</figcaption><span> In this figure, we find that the FI/Abs600 in device 1 is obviously higher than positive control,device 2,4 and 5. And another is that negative control and device 3,6 seems didn’t express GFP, or the expression is too low to observe.</span>>
</figure>
<h1 class="mdc-typography--display1">Discussion</h1>
<p class="mdc-typography--body2">
At first time, when we decide to transform plasmid into E.coli, what interesting is that we find device 1 can't growth in 170ug/ml or in 90ug/ml chloramphenicol at the same time we know exactly this plasmid is a high express one. Based on figure 3, we find that the growth curve of E.coli (except device 1) are all similar to the front part of S-Curve of Logistic regression, but it was strange that the two colony of device 1 seems don't change obviously in bacterial density which is different with other colonies. We conjectured this phenomenon came from the extremely high expression quantity of GFP which already produce severe cytotoxicity. As a result the growth of device 1 is very slow and can't growth in high concentration of chloramphenicol.
</p>
<p class="mdc-typography--body2">
In figure 4 and figure 5, we analyzed the relationship between fluorescence and Abs 600. Then find that the promotor of GFP in device 1 actually is strongest in 8 plasmid which as 5 times as positive control, device 2 and device 4. The expression of GFP in device 5 seems very low, only one-tenth of positive control.
</p>
<p class="mdc-typography--body2">
Also, we find that if the sampling time overlong, the E.coli growth will be restrain because of long time of low-temperature stress, in this suitcase, the growth of bacteria density is very slow and unparalleled in two colonies.
</p>
</article>
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[21.032, 33.667, 37.236, 46.199],
[6.658, 47.349, 85.106, 177.063],
[4.699, 43.932, 81.001, 166.369],
[0.471, 0.739, 1.218, 2.006],
[-0.029, 0.326, 1.478, 3.828],
[2.617, 47.176, 73.507, 110.345],
[2.350, 42.695, 69.011, 127.212],
[0.260, 7.932, 10.497, 15.538],
[0.431, 10.262, 12.250, 18.133],
[0.040, 0.233, 0.367, 0.936],
[-0.123, 0.133, 0.353, 0.842],
]
let chart_4 = new Highcharts.Chart('container-4', f34_config(f4));
chart_4.yAxis[0].setTitle({text: 'Fluorescence(AU)'})
// figure5
let f5 = [
[0.0045, 0.0701, 0.4178, 0.0842, 0.0132, 0.0494, 0.0059, -0.0012],
[0.0007, 0.0994, 0.5257, 0.0991, 0.0015, 0.1524, 0.0169, 0.0006],
[0.0004, 0.0763, 0.5048, 0.1085, 0.0014, 0.0901, 0.0113, 0.0004],
[0.0003, 0.0712, 0.4771, 0.1268, 0.0016, 0.0823, 0.0100, 0.0005],
]
let f5_error = [
[0.0027, 0.0042, 0.0278, 0.0047, 0.0093, 0.0048, 0.0037, 0.0036],
[0.0006, 0.0057, 0.0292, 0.0069, 0.0005, 0.0107, 0.0008, 0.0006],
[0.0003, 0.0027, 0.0256, 0.0031, 0.0003, 0.0036, 0.0005, 0.0002],
[0.0002, 0.0017, 0.0593, 0.0063, 0.0002, 0.0015, 0.0003, 0.0001]
]
function f5_config(f, f_e) {
let config = {
chart: {
zoomType: 'xy'
},
title: {
text: 'FI/Abs600(AU)'
},
xAxis: {
categories: [
'Negative Control',
'Positive Control',
'Test Device1',
'Test Device2',
'Test Device3',
'Test Device4',
'Test Device5',
'Test Device6',
]
},
yAxis: {
lineWidth: 1,
},
series: [{
type: 'column',
name: '0h'
},{
type: 'errorbar',
},{
type: 'column',
name: '2h'
},{
type: 'errorbar',
},{
type: 'column',
name: '4h',
yAxis: 0
},{
type: 'errorbar',
yAxis: 0
},{
type: 'column',
name: '6h'
},{
type: 'errorbar',
}]
}
for (var i = 0; i < f.length; i++) {
config.series[2*i].data = f[i];
let temp = []
for (var j = 0; j < f_e[i].length; j++) {
temp.push([ f[i][j]-f_e[i][j], f[i][j]+f_e[i][j] ]);
}
config.series[2*i+1].data = temp;
}
return config;
}
let chart_5 = new Highcharts.Chart('container-5', f5_config(f5, f5_error));
</script>
</body>
</html>