Difference between revisions of "Team:Hong Kong HKUST/InterLab"

 
(76 intermediate revisions by 6 users not shown)
Line 2: Line 2:
 
<html>
 
<html>
 
<head>
 
<head>
−
<link rel="stylesheet" href="https://maxcdn.bootstrapcdn.com/bootstrap/4.0.0/css/bootstrap.min.css" integrity="sha384-Gn5384xqQ1aoWXA+058RXPxPg6fy4IWvTNh0E263XmFcJlSAwiGgFAW/dAiS6JXm" crossorigin="anonymous">
+
 
<title>iGem HKUST 2018</title>
 
<title>iGem HKUST 2018</title>
 
<meta charset="utf-8" />
 
<meta charset="utf-8" />
 
<meta name="viewport" content="width=device-width, initial-scale=1" />
 
<meta name="viewport" content="width=device-width, initial-scale=1" />
−
<link rel="stylesheet" href="https://cdn.rawgit.com/logg926/hkustigem2018wikitemp/16f480d0/assets/css/main.css" />
+
 +
<link rel="stylesheet" type="text/css"
 +
href="https://2018.igem.org/wiki/index.php?title=Team:Hong_Kong_HKUST/Bootstrap.min.css&action=raw&ctype=text/css"/>
 +
<link rel="stylesheet" type="text/css"
 +
href="https://2018.igem.org/wiki/index.php?title=Team:Hong_Kong_HKUST/fontawesome.css&action=raw&ctype=text/css"/>
 +
<link rel="stylesheet" type="text/css"
 +
href="https://2018.igem.org/wiki/index.php?title=Template:Hong_Kong_HKUST/main.css&action=raw&ctype=text/css"/>
 +
<style>
 +
p{
 +
color:black;
 +
 
 +
}
 +
li {
 +
color:black;
 +
font-size:13pt;
 +
font-family:arial;
 +
}
 +
ol{
 +
color:black;
 +
font-size:13pt;
 +
font-family:arial;
 +
}
 +
figcaption{
 +
 
 +
color:black;
 +
font-size:13pt;
 +
font-family:arial;
 +
}
 +
</style>
 
</head>
 
</head>
 
<body style="background-color:black">
 
<body style="background-color:black">
 
<html>
 
<html>
 
<head>
 
<head>
−
<link rel="stylesheet" href="https://maxcdn.bootstrapcdn.com/bootstrap/4.0.0/css/bootstrap.min.css" integrity="sha384-Gn5384xqQ1aoWXA+058RXPxPg6fy4IWvTNh0E263XmFcJlSAwiGgFAW/dAiS6JXm" crossorigin="anonymous">
 
 
<title>Hielo by TEMPLATED</title>
 
<title>Hielo by TEMPLATED</title>
 
<meta charset="utf-8" />
 
<meta charset="utf-8" />
 
<meta name="viewport" content="width=device-width, initial-scale=1" />
 
<meta name="viewport" content="width=device-width, initial-scale=1" />
−
<link rel="stylesheet" href="assets/css/main.css" />
+
 
</head>
 
</head>
 
<body>
 
<body>
Line 23: Line 50:
 
<div class="inner">
 
<div class="inner">
 
<header class="align-center">
 
<header class="align-center">
−
<p>Eleifend vitae urna</p>
+
<img src="https://static.igem.org/mediawiki/2018/3/32/T--Hong_Kong_HKUST--Interlabbanner.png" class="rounded mx-auto d-block" alt="..." width="100%" height="100%">
−
<h2>Generic Page Template</h2>
+
 
</header>
 
</header>
 
</div>
 
</div>
Line 30: Line 57:
  
  
−
   <img src="https://static.igem.org/mediawiki/2018/d/d3/T--Hong_Kong_HKUST--_PEkachu-Picturelogooeijne1.png" class="rounded mx-auto d-block" alt="...">
+
    
  
  
Line 39: Line 66:
 
<div class="content">
 
<div class="content">
 
<header class="align-center">
 
<header class="align-center">
−
<p align = "center">maecenas sapien feugiat ex purus</p>
+
−
<h2>Lorem ipsum dolor</h2>
+
 
</header>
 
</header>
−
<p>Lorem ipsum dolor sit amet, consectetur adipiscing elit. Cras at dignissim augue, in iaculis neque. Etiam bibendum felis ac vulputate pellentesque. Cras non blandit quam. Nunc porta, est non posuere sagittis, neque nunc pellentesque diam, a iaculis lacus urna vitae purus. In non dui vel est tempor faucibus. Aliquam erat volutpat. Quisque vel est vitae nibh laoreet auctor. In nec libero dui. Nulla ullamcorper, dolor nec accumsan viverra, libero eros rutrum metus, vel lacinia magna odio non nunc. Praesent semper felis eu rhoncus aliquam. Donec at quam ac libero vestibulum pretium. Nunc faucibus vel arcu in malesuada. Aenean at velit odio. Vestibulum ante ipsum primis in faucibus orci luctus et ultrices posuere cubilia Curae; Maecenas commodo erat eget molestie sollicitudin. Donec imperdiet, ex sed blandit dictum, ipsum metus ultrices arcu, vitae euismod nisl sapien vitae tortor.</p>
+
<h2>OUR INTERLAB OBJECTIVES</h2>
 +
<p>The iGEM Interlab 2018 aims to reduce lab-to-lab variability in fluorescence measurements that were shown in previous interlab studies which use an optical density (O.D.) as the normalization method of fluorescence. Since O.D. is an approximation of cell number, the interlab this year attempts to address the problem by two orthogonal approaches. Hypothesized that silica beads have similar light scattering properties as the cells due to their similarities in size and shape, one of the approaches is to convert the absorbance of cells to the absorbance of a known concentration of silica beads <sup>[1]</sup>. Adopting a more direct normalization method, the other approach is to normalize the absorbance of cells by absolute cell counts or colony-forming units (CFU).   </p>
  
−
<p>Vivamus nec odio ac ligula congue feugiat at vitae leo. Aenean sem justo, finibus sed dui eu, accumsan facilisis dolor. Fusce quis dui eget odio iaculis aliquam vel sed velit. Nulla pellentesque posuere semper. Nulla eu sagittis lorem, a auctor nulla. Sed ac condimentum orci, ac varius ante. Nunc blandit quam sit amet sollicitudin sodales.</p>
+
<h2>Method:</h2>
  
−
<p>Vivamus ultricies mollis mauris quis molestie. Quisque eu mi velit. In et cursus nibh. Donec facilisis, orci sed mollis hendrerit, nunc risus mattis odio, eget efficitur nisl orci a lectus. Aenean finibus neque convallis orci sollicitudin tincidunt. Vivamus lacinia facilisis diam, quis facilisis nisi luctus nec. Aliquam ac molestie enim, ut ultrices elit. Fusce laoreet vulputate risus in tincidunt. Sed commodo mollis maximus. Nullam varius laoreet nibh sit amet facilisis. Donec ac odio vehicula, consequat elit et, sodales justo. Vestibulum ante ipsum primis in faucibus orci luctus et ultrices posuere cubilia Curae; Nullam ac auctor mauris, in hendrerit libero. </p>
+
<p>All procedures were performed according to the given iGEM protocol <sup>[2]</sup>, except that the O.D. measurement setting was changed from OD<sub>600</sub> to OD<sub>595</sub>, due to the limited options of plate reader in HKUST. After further discussion with the iGEM headquarter, we retained the data to be OD<sub>595</sub>.
 +
</p>
 +
<h2>Machines, materials and parts:</h2>
 +
<h3><i>Machines:</i></h3><br/>
 +
<ul style="color:black;
 +
font-size:13pt;
 +
font-family:arial;">
 +
<li><p>Envision Multilabel Reader (Model: EnVision Xcite)</p></li> <br/>
 +
</ul>
 +
<p>
 +
*To know more about the setting of EnVision multilabel reader, please <a href="">click</a> <br/>
 +
</p>
 +
<p>
 +
<!--<ul style="color:black;
 +
font-size:13pt;
 +
font-family:arial;">-->
 +
<h3><i>Materials:</i></h3><br/>
 +
<li><b>LUDOX CL-X</b>: 45% colloidal silica suspension, used as single reference point for converting absorbance (Abs<sub>600</sub>) to OD<sub>600</sub> <br/>.
 +
</li>
 +
<li><b>Silica beads</b>: Microsphere suspension that mimics the shape and size of typical <i>E.coli</i> cell. With known concentration, it can be used for the conversion of absorbance measurement to the universal standard concentration of bead measurement.
 +
 
 +
</li> <br/>
 +
<li><b>Fluorescein</b>: Sodium fluorescein was used for obtaining the standard fluorescence curve.</b>
 +
</li> <br/>
 +
<li><i>E.coli</i> strain DH5αCompetent cell: used for transformation, the protocol used for making it can view in <a href="http://www.unc.edu/depts/marzluff/Marzluff/Protocols_files/Inoue%20Method%20for%20Preparation%20of%20Ultracompetent%20cells.pdf">here</a>
 +
</li> <br/>
 +
 
 +
<!--</ul>-->
 +
</p>
 +
 
 +
<h3><i>Parts:</i></h3><br/>
 +
<table style="width:100% ;color:black;
 +
font-size:13pt;
 +
font-family:arial;">
 +
  <tr>
 +
    <th>Parts</th>
 +
    <th>Parts location on the kits plate</th>
 +
    <th>Parts used as the promoter(strength)</th>
 +
<th>Parts used as the RBS(Efficiency)</th>
 +
<th>Reporter Gene</th>
 +
<th>Parts used as the Terminator</th>
 +
  </tr>
 +
  <tr>
 +
    <td>Positive Control(BBa_I20270)</td>
 +
    <td>Plate 7 Well 2B</td>
 +
    <td>BBa_J23151 (nil)</td>
 +
    <td>BBa_B0032 (0.3)</td>
 +
    <td>GFP</td>
 +
    <td>BBa_B0010, BBa_B0012</td>
 +
  </tr>
 +
  <tr>
 +
    <td>Negative Control (BBa_R0040)</td>
 +
    <td>Plate 7 Well 2D</td>
 +
    <td>BBa_R0040 (nil)</td>
 +
    <td>nil</td>
 +
    <td>GFP</td>
 +
<td>BBa_B0010, BBa_B0012</td>
 +
  </tr>
 +
  <tr>
 +
    <td>Test Device 1 (BBa_J364000)</td>
 +
    <td>Plate 7 Well 2F</td>
 +
    <td>BBa_J23101 (1791au)</td>
 +
    <td>BBa_B0034 (1.0)</td>
 +
    <td>GFP</td>
 +
<td>BBa_B0010, BBa_B0012</td>
 +
  </tr>
 +
  <tr>
 +
    <td>Test Device 2 (BBa_J364001)</td>
 +
    <td>Plate 7 Well 2H</td>
 +
    <td>BBa_J23106 (1185au)</td>
 +
    <td>BBa_B0034 (1.0)</td>
 +
    <td>GFP</td>
 +
<td>BBa_B0010, BBa_B0012</td>
 +
  </tr>
 +
  <tr>
 +
    <td>Test Device 3 (BBa_J364002)</td>
 +
    <td>Plate 7 Well 2J</td>
 +
    <td>BBa_J23117 (162au)</td>
 +
    <td>BBa_B0034 (1.0)</td>
 +
    <td>GFP</td>
 +
<td>BBa_B0010, BBa_B0012</td>
 +
  </tr>
 +
  <tr>
 +
    <td>Test Device 4 (BBa_J364007)</td>
 +
    <td>Plate 7 Well 2L</td>
 +
    <td>BBa_J23100(2547au)</td>
 +
    <td>BBa_B0034* (nil)</td>
 +
    <td>GFP</td> 
 +
<td>BBa_B0010, BBa_B0012</td>
 +
  </tr>
 +
  <tr>
 +
  <td>Test Device 4 (BBa_J364007)</td>
 +
  <td>Plate 7 Well 2L</td>
 +
  <td>BBa_J23100(2547au)</td>
 +
  <td>BBa_B0034* (nil)</td>
 +
  <td>GFP</td>
 +
<td>BBa_B0010, BBa_B0012</td>
 +
  </tr>
 +
</table>
 +
</p>
 +
<h2>Result:</h2>
 +
<p>
 +
<h3><i>Calibrations:</i></h3>
 +
</p>
 +
<p>
 +
Conversion factor of OD<sub>600</sub>(OD<sub>600</sub>/Abs<sub>600</sub>) = 3.036
 +
 
 +
<br>
 +
<caption style="text-align:center;color:black;
 +
font-size:13pt;
 +
font-family:arial;">Table 2: Conversion factor calculation</caption> </p>
 +
<table style="width:100%;color:black;
 +
font-size:13pt;
 +
font-family:arial;">
 +
  <tr>
 +
    <th>  </th>
 +
    <th>LUDOX CL-X</th>
 +
    <th>H<sub>2</sub>0</th>
 +
 
 +
  </tr>
 +
  <tr>
 +
    <td>Replicate 1</td>
 +
    <td>0.045</td>
 +
    <td>0.024</td>
 +
 
 +
  </tr>
 +
  <tr>
 +
    <td>Replicate 2</td>
 +
    <td>0.045</td>
 +
    <td>0.025</td>
 +
  </tr>
 +
  <tr>
 +
    <td>Replicate 3</td>
 +
    <td>0.044</td>
 +
    <td>0.024</td>
 +
  </tr>
 +
  <tr>
 +
    <td>Replicate 4</td>
 +
    <td>0.049</td>
 +
    <td>0.027</td>
 +
  </tr>
 +
  <tr>
 +
    <td>Arithmethic mean</td>
 +
    <td>0.046</td>
 +
    <td>0.025</td>
 +
  </tr>
 +
  <tr>
 +
    <td>Corrected Abs<sub>600</sub></td>
 +
    <td>0.021</td>
 +
   
 +
  </tr>
 +
  <tr>
 +
  <td>Reference  OD<sub>600</sub></td>
 +
  <td>0.063</td> 
 +
  </tr>
 +
  <tr>
 +
    <td>OD<sub>600</sub>/Abs<sub>600</sub></td>
 +
    <td>3.036</td>
 +
  </tr>
 +
</table>
 +
</p>
 +
<figure>
 +
<center><img src="https://static.igem.org/mediawiki/2018/1/1f/T--Hong_Kong_HKUST--Particlestandardnew.png" class="img-fluid" alt="Responsive image" width="500px" height="500px" ></center>
 +
<center><figcaption style="color:black;
 +
font-size:13pt;
 +
font-family:arial;"><b>Fig. 2a</b> Particle Standard Curve</figcaption></center>
 +
<br>
 +
</figure>
 +
 
 +
<figure>
 +
<center><img src="https://static.igem.org/mediawiki/2018/2/23/T--Hong_Kong_HKUST--Particlestandardcurvelog.png" class="img-fluid" alt="Responsive image" width="500px" height="500px"></center>
 +
<center><figcaption style="color:black;
 +
font-size:13pt;
 +
font-family:arial;"><b>Fig.2b</b> Particle Standard Curve (log scale)
 +
</figcaption></center>
 +
<br>
 +
</figure>
 +
 
 +
<figure>
 +
<center><img src="https://static.igem.org/mediawiki/2018/b/b7/T--Hong_Kong_HKUST--FLuorescein_standard_curve%28new%29.png" class="img-fluid" alt="Responsive image" width="500px" height="500px"></center>
 +
<center><figcaption style="color:black;
 +
font-size:13pt;
 +
font-family:arial;"><b>Fig.3a</b> Fluorescein standard curve</figcaption></center>
 +
</figure>
 +
<br>
 +
 
 +
<figure>
 +
<center><img src="https://static.igem.org/mediawiki/2018/0/0a/T--Hong_Kong_HKUST--Fluoresceinlog.png" class="img-fluid" alt="Responsive image" width="500px" height="500px"></center>
 +
<center><figcaption style="color:black;
 +
font-size:13pt;
 +
font-family:arial;"><b>Fig.3b</b> Fluorescein standard curve (log scale)
 +
</br>
 +
The non-linear fluorescence standard curve is conjectured to be a result of detector over-saturation. </br>This could be inferred from a linear curve at low concentrations of fluorescein while reaching plateau at high concentrations.
 +
</figcaption></center>
 +
</figure>
 +
<br>
 +
<p> </p>
 +
 
 +
<h2>Conversion of absorbance of cells to absorbance of a known concentration of beads.</h2>
 +
<br/>
 +
<figure>
 +
<center><img src="https://static.igem.org/mediawiki/2018/d/d2/T--Hong_Kong_HKUST--AverageuMInterlabwiki%28new%29.png" class="img-fluid" alt="Responsive image" width="500px" height="500px"></center>
 +
<center><figcaption style="color:black;
 +
font-size:13pt;
 +
font-family:arial;"><b>Fig.4a</b> Average <sub>u</sub>M Fluorescein / OD<sub>600</sub> of each devices
 +
</figcaption></center>
 +
</figure>
 +
<br>
 +
 
 +
<figure>
 +
<center><img src="https://static.igem.org/mediawiki/2018/2/27/T--Hong_Kong_HKUST--AverageMEFLInterlabwiki.png" class="img-fluid" alt="Responsive image" width="500px" height="500px"></center>
 +
<center><figcaption style="color:black;
 +
font-size:13pt;
 +
font-family:arial;"><b>Fig.4b</b> Fluorescein standard curve (log scale)
 +
</figcaption></center>
 +
</figure>
 +
<br>
 +
<h2>Counting colony-forming units (CFUs) from the sample
 +
</h2><br/>
 +
 
 +
<p>
 +
Colonies count: <br/>
 +
Negative control (BBa_R0040):
 +
</p>
 +
<table style="color:black;
 +
font-size:13pt;
 +
font-family:arial;">
 +
<tr>
 +
<th></th>
 +
<th>Dillution 3</th>
 +
<th>Dillution 4</th>
 +
<th>Dillution 5</th>
 +
</tr>
 +
<tr>
 +
<td>Colony 1, Replicate 1</td>
 +
<td>180</td>
 +
<td>13</td>
 +
<td>3</td>
 +
</tr>
 +
<tr>
 +
<td>Colony 1, Replicate 2</td>
 +
<td>120</td>
 +
<td>14</td>
 +
<td>3</td>
 +
</tr>
 +
<tr>
 +
<td>Colony 1, Replicate 3</td>
 +
<td>197</td>
 +
<td>33</td>
 +
<td>2</td>
 +
</tr>
 +
<tr>
 +
<td>Colony 2, Replicate 1</td>
 +
<td>283</td>
 +
<td>33</td>
 +
<td>2</td>
 +
</tr>
 +
<tr>
 +
<td>Colony 2, Replicate 2</td>
 +
<td>214</td>
 +
<td>28</td>
 +
<td>3</td>
 +
</tr>
 +
<tr>
 +
<td>Colony 2, Replicate 3</td>
 +
<td>218</td>
 +
<td>29</td>
 +
<td>1</td>
 +
</tr>
 +
</table>
 +
<p>
 +
Positive control ((BBa_I120270):
 +
</p>
 +
<table style="color:black;
 +
font-size:13pt;
 +
font-family:arial;">
 +
<tr>
 +
<th></th>
 +
<th>Dillution 3</th>
 +
<th>Dillution 4</th>
 +
<th>Dillution 5</th>
 +
</tr>
 +
<tr>
 +
<td>Colony 1, Replicate 1</td>
 +
<td>228</td>
 +
<td>29</td>
 +
<td>1</td>
 +
</tr>
 +
<tr>
 +
<td>Colony 1, Replicate 2</td>
 +
<td>184</td>
 +
<td>25</td>
 +
<td>1</td>
 +
</tr>
 +
<tr>
 +
<td>Colony 1, Replicate 3</td>
 +
<td>153</td>
 +
<td>25</td>
 +
<td>1</td>
 +
</tr>
 +
<tr>
 +
<td>Colony 2, Replicate 1</td>
 +
<td>254</td>
 +
<td>19</td>
 +
<td>3</td>
 +
</tr>
 +
<tr>
 +
<td>Colony 2, Replicate 2</td>
 +
<td>168</td>
 +
<td>27</td>
 +
<td>2</td>
 +
</tr>
 +
<tr>
 +
<td>Colony 2, Replicate 3</td>
 +
<td>213</td>
 +
<td>24</td>
 +
<td>3</td>
 +
</tr>
 +
</table>
 +
 
 +
<p>
 +
Colony-forming unit (CFU):
 +
Negative control (BBa_R0040):
 +
</p>
 +
<table style="color:black;
 +
font-size:13pt;
 +
font-family:arial;">
 +
<tr>
 +
<th></th>
 +
<th>Dillution 3</th>
 +
<th>Dillution 4</th>
 +
<th>Dillution 5</th>
 +
</tr>
 +
<tr>
 +
<td>Colony 1, Replicate 1</td>
 +
<td>1.44E+07</td>
 +
<td>1.04E+07</td>
 +
<td>2.40E+07</td>
 +
</tr>
 +
<tr>
 +
<td>Colony 1, Replicate 2</td>
 +
<td>9.60E+06</td>
 +
<td>1.12E+07</td>
 +
<td>2.40E+07</td>
 +
</tr>
 +
<tr>
 +
<td>Colony 1, Replicate 3</td>
 +
<td>1.58E+07</td>
 +
<td>2.64E+07</td>
 +
<td>1.60E+07</td>
 +
</tr>
 +
<tr>
 +
<td>Colony 2, Replicate 1</td>
 +
<td>2.26E+07</td>
 +
<td>1.84E+07</td>
 +
<td>1.60E+07</td>
 +
</tr>
 +
<tr>
 +
<td>Colony 2, Replicate 2</td>
 +
<td>1.71E+07</td>
 +
<td>2.24E+07</td>
 +
<td>2.40E+07</td>
 +
</tr>
 +
<tr>
 +
<td>Colony 2, Replicate 3</td>
 +
<td>1.74E+07</td>
 +
<td>2.32E+07</td>
 +
<td>8.00E+06</td>
 +
</tr>
 +
</table>
 +
<h2>
 +
Average </h2>
 +
<ul style="color:black;
 +
font-size:13pt;
 +
font-family:arial;">
 +
<li>Colony 1: 1.69E+07 CFU/ml/0.1OD</li>
 +
<li>Colony 2: 1.88E+07 CFU/ml/0.1OD</li>
 +
<li>Average: 1.785E+07 CFU/ml/0.1OD</li>
 +
<li>Using conversion factor OD/Abs= 3.036</li>
 +
<li>Conversion factor: CFU/Abs/ml= 54.34 CFU/Abs/ml</li>
 +
</ul>
 +
 
 +
</p>
 +
<h2>Conclusion:</h2>
 +
<p>
 +
There is no significant difference in the pattern of normalized fluorescence values between using O.D. and particle count, as illustrated in Figure 4 and 5. The normalized fluorescence values of the devices are consistent with their respective promoter strengths, with device 1 (BBa_J23101) as the highest fluorescence value (i.e. 1791 a.u.) and device 3 (BBa_J23117) as the lowest fluorescence value (i.e. 162 a.u.). However, cell quantification by colony-forming units failed to reproduce the modeled cell concentration by silica beads. This may conclude that the two methods, CFU cell count and silica beads, may not be able to produce a consistent value of cell concentration.   
 +
</p>
 +
<section id="One" class="wrapper style3">
 +
<div class="inner">
 +
<header class="align-center">
 +
 +
<h2>REFERENCES:</h2>
 +
 
 +
</header>
 +
</div>
 +
</section>
 +
<p>1. Measurement/InterLab - 2018.igem.org", 2018.igem.org, 2018. Available: https://2018.igem.org/Measurement/InterLab
 +
</p>
 +
<p>2. InterLab Plate Reader Protocol. The 2018 International Genetically Engineered Machine. Available:https://static.igem.org/mediawiki/2018/0/09/2018_InterLab_Plate_Reader_Protocol.pdf
 +
</p>
 
</div>
 
</div>
 
</div>
 
</div>
Line 59: Line 485:
  
 
<!-- One -->
 
<!-- One -->
−
<section id="One" class="wrapper style3">
+
−
<div class="inner">
+
−
<header class="align-center">
+
−
<p>Eleifend vitae urna</p>
+
−
<h2>Generic Page Template</h2>
+
−
 
+
−
</header>
+
−
</div>
+
−
</section>
+
  
  
 
 
−
<!-- Two -->
+
−
<section id="two" class="wrapper style2">
+
−
<div class="inner">
+
−
<div class="box">
+
−
<div class="content">
+
−
<header class="align-center">
+
−
<p>maecenas sapien feugiat ex purus</p>
+
−
<h2>Lorem ipsum dolor</h2>
+
−
</header>
+
−
 
+
−
<img src="https://static.igem.org/mediawiki/2018/4/44/T--Hong_Kong_HKUST--_candidphoto-6.jpeg" class="img-fluid" alt="Responsive image">
+
−
 
+
−
<p>Lorem ipsum dolor sit amet, consectetur adipiscing elit. Cras at dignissim augue, in iaculis neque. Etiam bibendum felis ac vulputate pellentesque. Cras non blandit quam. Nunc porta, est non posuere sagittis, neque nunc pellentesque diam, a iaculis lacus urna vitae purus. In non dui vel est tempor faucibus. Aliquam erat volutpat. Quisque vel est vitae nibh laoreet auctor. In nec libero dui. Nulla ullamcorper, dolor nec accumsan viverra, libero eros rutrum metus, vel lacinia magna odio non nunc. Praesent semper felis eu rhoncus aliquam. Donec at quam ac libero vestibulum pretium. Nunc faucibus vel arcu in malesuada. Aenean at velit odio. Vestibulum ante ipsum primis in faucibus orci luctus et ultrices posuere cubilia Curae; Maecenas commodo erat eget molestie sollicitudin. Donec imperdiet, ex sed blandit dictum, ipsum metus ultrices arcu, vitae euismod nisl sapien vitae tortor.</p>
+
−
+
−
<p>Vivamus nec odio ac ligula congue feugiat at vitae leo. Aenean sem justo, finibus sed dui eu, accumsan facilisis dolor. Fusce quis dui eget odio iaculis aliquam vel sed velit. Nulla pellentesque posuere semper. Nulla eu sagittis lorem, a auctor nulla. Sed ac condimentum orci, ac varius ante. Nunc blandit quam sit amet sollicitudin sodales.</p>
+
−
+
−
<p>Vivamus ultricies mollis mauris quis molestie. Quisque eu mi velit. In et cursus nibh. Donec facilisis, orci sed mollis hendrerit, nunc risus mattis odio, eget efficitur nisl orci a lectus. Aenean finibus neque convallis orci sollicitudin tincidunt. Vivamus lacinia facilisis diam, quis facilisis nisi luctus nec. Aliquam ac molestie enim, ut ultrices elit. Fusce laoreet vulputate risus in tincidunt. Sed commodo mollis maximus. Nullam varius laoreet nibh sit amet facilisis. Donec ac odio vehicula, consequat elit et, sodales justo. Vestibulum ante ipsum primis in faucibus orci luctus et ultrices posuere cubilia Curae; Nullam ac auctor mauris, in hendrerit libero. </p>
+
−
</div>
+
−
</div>
+
−
</div>
+
−
</section>
+
  
 
 
  
 
<!-- Scripts -->
 
<!-- Scripts -->
−
<script src="https://cdn.rawgit.com/logg926/hkustigem2018wikitemp/16f480d0/assets/js/jquery.min.js"></script>
+
<script type="text/javascript" src="https://2018.igem.org/wiki/index.php?title=Team:Hong_Kong_HKUST/jquery.min.js/&action=raw&ctype=text/javascript"></script>
−
<script src="https://cdn.rawgit.com/logg926/hkustigem2018wikitemp/16f480d0/assets/js/jquery.scrollex.min.js"></script>
+
<script type="text/javascript" src="https://2018.igem.org/wiki/index.php?title=Team:Hong_Kong_HKUST/jquery.scrollex.min.js/&action=raw&ctype=text/javascript"></script>
−
<script src="https://cdn.rawgit.com/logg926/hkustigem2018wikitemp/16f480d0/assets/js/skel.min.js"></script>
+
<script type="text/javascript" src="https://2018.igem.org/wiki/index.php?title=Team:Hong_Kong_HKUST/skel.min.js/&action=raw&ctype=text/javascript"></script>
−
<script src="https://cdn.rawgit.com/logg926/hkustigem2018wikitemp/16f480d0/assets/js/util.js"></script>
+
<script type="text/javascript" src="https://2018.igem.org/wiki/index.php?title=Team:Hong_Kong_HKUST/util.js/&action=raw&ctype=text/javascript"></script>
−
<script src="https://cdn.rawgit.com/logg926/hkustigem2018wikitemp/16f480d0/assets/js/main.js"></script>
+
<script type="text/javascript" src="https://2018.igem.org/wiki/index.php?title=Team:Hong_Kong_HKUST/main.js/&action=raw&ctype=text/javascript"></script>  
 
</body>
 
</body>
 
</html>
 
</html>
 
{{ Hong_Kong_HKUST/footer }}
 
{{ Hong_Kong_HKUST/footer }}

Latest revision as of 18:23, 17 October 2018

iGem HKUST 2018 Hielo by TEMPLATED
...

OUR INTERLAB OBJECTIVES

The iGEM Interlab 2018 aims to reduce lab-to-lab variability in fluorescence measurements that were shown in previous interlab studies which use an optical density (O.D.) as the normalization method of fluorescence. Since O.D. is an approximation of cell number, the interlab this year attempts to address the problem by two orthogonal approaches. Hypothesized that silica beads have similar light scattering properties as the cells due to their similarities in size and shape, one of the approaches is to convert the absorbance of cells to the absorbance of a known concentration of silica beads [1]. Adopting a more direct normalization method, the other approach is to normalize the absorbance of cells by absolute cell counts or colony-forming units (CFU).

Method:

All procedures were performed according to the given iGEM protocol [2], except that the O.D. measurement setting was changed from OD600 to OD595, due to the limited options of plate reader in HKUST. After further discussion with the iGEM headquarter, we retained the data to be OD595.

Machines, materials and parts:

Machines:


  • Envision Multilabel Reader (Model: EnVision Xcite)


*To know more about the setting of EnVision multilabel reader, please click

Materials:


  • LUDOX CL-X: 45% colloidal silica suspension, used as single reference point for converting absorbance (Abs600) to OD600
    .
  • Silica beads: Microsphere suspension that mimics the shape and size of typical E.coli cell. With known concentration, it can be used for the conversion of absorbance measurement to the universal standard concentration of bead measurement.

  • Fluorescein: Sodium fluorescein was used for obtaining the standard fluorescence curve.

  • E.coli strain DH5αCompetent cell: used for transformation, the protocol used for making it can view in here

    • Parts:


    Parts Parts location on the kits plate Parts used as the promoter(strength) Parts used as the RBS(Efficiency) Reporter Gene Parts used as the Terminator
    Positive Control(BBa_I20270) Plate 7 Well 2B BBa_J23151 (nil) BBa_B0032 (0.3) GFP BBa_B0010, BBa_B0012
    Negative Control (BBa_R0040) Plate 7 Well 2D BBa_R0040 (nil) nil GFP BBa_B0010, BBa_B0012
    Test Device 1 (BBa_J364000) Plate 7 Well 2F BBa_J23101 (1791au) BBa_B0034 (1.0) GFP BBa_B0010, BBa_B0012
    Test Device 2 (BBa_J364001) Plate 7 Well 2H BBa_J23106 (1185au) BBa_B0034 (1.0) GFP BBa_B0010, BBa_B0012
    Test Device 3 (BBa_J364002) Plate 7 Well 2J BBa_J23117 (162au) BBa_B0034 (1.0) GFP BBa_B0010, BBa_B0012
    Test Device 4 (BBa_J364007) Plate 7 Well 2L BBa_J23100(2547au) BBa_B0034* (nil) GFP BBa_B0010, BBa_B0012
    Test Device 4 (BBa_J364007) Plate 7 Well 2L BBa_J23100(2547au) BBa_B0034* (nil) GFP BBa_B0010, BBa_B0012

    Result:

    Calibrations:

    Conversion factor of OD600(OD600/Abs600) = 3.036
    Table 2: Conversion factor calculation

    LUDOX CL-X H20
    Replicate 1 0.045 0.024
    Replicate 2 0.045 0.025
    Replicate 3 0.044 0.024
    Replicate 4 0.049 0.027
    Arithmethic mean 0.046 0.025
    Corrected Abs600 0.021
    Reference OD600 0.063
    OD600/Abs600 3.036

    Responsive image
    Fig. 2a Particle Standard Curve

    Responsive image
    Fig.2b Particle Standard Curve (log scale)

    Responsive image
    Fig.3a Fluorescein standard curve

    Responsive image
    Fig.3b Fluorescein standard curve (log scale)
    The non-linear fluorescence standard curve is conjectured to be a result of detector over-saturation.
    This could be inferred from a linear curve at low concentrations of fluorescein while reaching plateau at high concentrations.

    Conversion of absorbance of cells to absorbance of a known concentration of beads.


    Responsive image
    Fig.4a Average uM Fluorescein / OD600 of each devices

    Responsive image
    Fig.4b Fluorescein standard curve (log scale)

    Counting colony-forming units (CFUs) from the sample


    Colonies count:
    Negative control (BBa_R0040):

    Dillution 3 Dillution 4 Dillution 5
    Colony 1, Replicate 1 180 13 3
    Colony 1, Replicate 2 120 14 3
    Colony 1, Replicate 3 197 33 2
    Colony 2, Replicate 1 283 33 2
    Colony 2, Replicate 2 214 28 3
    Colony 2, Replicate 3 218 29 1

    Positive control ((BBa_I120270):

    Dillution 3 Dillution 4 Dillution 5
    Colony 1, Replicate 1 228 29 1
    Colony 1, Replicate 2 184 25 1
    Colony 1, Replicate 3 153 25 1
    Colony 2, Replicate 1 254 19 3
    Colony 2, Replicate 2 168 27 2
    Colony 2, Replicate 3 213 24 3

    Colony-forming unit (CFU): Negative control (BBa_R0040):

    Dillution 3 Dillution 4 Dillution 5
    Colony 1, Replicate 1 1.44E+07 1.04E+07 2.40E+07
    Colony 1, Replicate 2 9.60E+06 1.12E+07 2.40E+07
    Colony 1, Replicate 3 1.58E+07 2.64E+07 1.60E+07
    Colony 2, Replicate 1 2.26E+07 1.84E+07 1.60E+07
    Colony 2, Replicate 2 1.71E+07 2.24E+07 2.40E+07
    Colony 2, Replicate 3 1.74E+07 2.32E+07 8.00E+06

    Average

    • Colony 1: 1.69E+07 CFU/ml/0.1OD
    • Colony 2: 1.88E+07 CFU/ml/0.1OD
    • Average: 1.785E+07 CFU/ml/0.1OD
    • Using conversion factor OD/Abs= 3.036
    • Conversion factor: CFU/Abs/ml= 54.34 CFU/Abs/ml

    Conclusion:

    There is no significant difference in the pattern of normalized fluorescence values between using O.D. and particle count, as illustrated in Figure 4 and 5. The normalized fluorescence values of the devices are consistent with their respective promoter strengths, with device 1 (BBa_J23101) as the highest fluorescence value (i.e. 1791 a.u.) and device 3 (BBa_J23117) as the lowest fluorescence value (i.e. 162 a.u.). However, cell quantification by colony-forming units failed to reproduce the modeled cell concentration by silica beads. This may conclude that the two methods, CFU cell count and silica beads, may not be able to produce a consistent value of cell concentration.

    REFERENCES:

    1. Measurement/InterLab - 2018.igem.org", 2018.igem.org, 2018. Available: https://2018.igem.org/Measurement/InterLab

    2. InterLab Plate Reader Protocol. The 2018 International Genetically Engineered Machine. Available:https://static.igem.org/mediawiki/2018/0/09/2018_InterLab_Plate_Reader_Protocol.pdf

    ©IGEM HKUST 2018. All rights reserved.