Difference between revisions of "Team:NKU CHINA/interlab"

Latest revision as of 07:27, 1 October 2018

Overview

Poverty in taking reliable and repeatable measurements remains a key obstacle in establishing synthetic biology as an engineering discipline. The Measurement Committee has been studying the measurement procedure for green fluorescent protein (GFP) over the last several years by interlab. The most commonly used markers though GFP is in synthetic biology, labs often resort to making relative comparisons, which makes it difficult for labs to share and data and/or constructs.

The goal of the fifth iGEM InterLab Study is to identify and correct the sources of systematic variability in synthetic biology measurements by answering the question "Can we reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of OD? "

As we know in the previous study, the fluorescence value measured by a plate reader is an aggregate measurement of an entire population of cells, we need to divide the total fluorescence by the number of cells in order to determine the mean expression level of GFP per cell. Due to the fact that the "optical density (OD)" of the sample is an approximation of the number of cells varying from lab to lab, we decided to use a special silica beads that are roughly the same size and shape as a typical E. coli cell to set up a universal, standard "equivalent concentration of beads" measurement.

Materials

Reagents and Apparatus

Competent cells (Escherichia coli strain DH5α)
1 mL LUDOX CL-X (provided in kit)
300 µL Silica beads - Microsphere suspension (provided in kit, 4.7 x 10⁸ microspheres)
LB (Luria Bertani) media
Fluorescein (provided in kit)
10 mL 1xPBS pH 7.4-7.6 (phosphate buffered saline; provided by team)
Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH)
50 mL Falcon tube (or equivalent, preferably amber or covered in foil to block light)
Incubator at 37°C
1.5 mL eppendorf tubes for sample storage
Ice bucket with ice
Micropipettes and tips
96 well plate, black with clear flat bottom preferred (provided by team)

Devices

From Distribution Kit, all in pSB1C3 backbone:

Negative control BBa_R0040
Positive control BBa_I20270
Test Device 1 BBa_J364000
Test Device 2 BBa_J364001
Test Device 3 BBa_J364002
Test Device 4 BBa_J364007
Test Device 5 BBa_J364008
Test Device 6 BBa_J364009

Methods

OD₆₀₀ Reference Point

Add 100 µL LUDOX into wells A1, B1, C1, D1
Add 100 µL of dd H₂O into wells A2, B2, C2, D2
Measure absorbance at 600 nm of all samples in the measurement mode you plan to use for cell measurements
Record the data in the table below or in your notebook
Import data into Excel sheet provided (OD₆₀₀ reference point tab)

Particle Standard Curve

Obtain the tube labeled "Silica Beads" from the InterLab test kit and vortex vigorously for 30 seconds
Immediately pipet 96 µL microspheres into a 1.5 mL eppendorf tube
Add 904 µL of ddH₂O to the microspheres
Vortex well. This is your Microsphere Stock Solution
Repeat dilution series for rows B, C, D
Re-Mix (Pipette up and down) each row of plate immediately before putting in the plate reader
Measure Abs₆₀₀ of all samples in instrument
Record the data in your notebook
Import data into Excel sheet provided (particle standard curve tab)

Fluorescence Standard Curve

Spin down fluorescein kit tube to make sure pellet is at the bottom of tube.
Prepare 10x fluorescein stock solution (100 µM) by resuspending fluorescein in 1 mL of 1xPBS
Dilute the 10x fluorescein stock solution with 1xPBS to make a 1x fluorescein solution with concentration 10 µM: 100 µL of 10x fluorescein stock into 900 µL 1xPBS
Add 100 µL of PBS into wells A2, B2, C2, D2...A12, B12, C12, D12
Add 200 µL of fluorescein 1x stock solution into A1, B1, C1, D1
Transfer 100 µL of fluorescein stock solution from A1 into A2
Mix A2 by pipetting up and down 3x and transfer 100 µL into A3...
Mix A3 by pipetting up and down 3x and transfer 100 µL into A4...
Mix A4 by pipetting up and down 3x and transfer 100 µL into A5...
Mix A5 by pipetting up and down 3x and transfer 100 µL into A6...
Mix A6 by pipetting up and down 3x and transfer 100 µL into A7...
Mix A7 by pipetting up and down 3x and transfer 100 µL into A8...
Mix A8 by pipetting up and down 3x and transfer 100 µL into A9...
Mix A9 by pipetting up and down 3x and transfer 100 µL into A10...
Mix A10 by pipetting up and down 3x and transfer 100 µL into A11...
Mix A11 by pipetting up and down 3x and transfer 100 µL into liquid waste
Repeat dilution series for rows B, C, D
Measure fluorescence of all samples in instrument
Record the data in your notebook
Import data into Excel sheet provided (fluorescein standard curve tab)

Competent Cells and Transformation

Resuspend DNA in selected wells in the Distribution Kit with 10 &#181L dH₂O
Thaw competent cells on ice
Pipette 50 µL of competent cells into 1.5 mL tube
Pipette 1 µL of resuspended DNA into 1.5 mL tube
Pipette 1 µL of control DNA into 2 mL tube
Close 1.5 mL tubes, incubate on ice for 30 min
Heat shock tubes at 42°C for 45 sec
Incubate on ice for 5 min
Pipette 950 µL SOC media to each transformation
Incubate at 37°C for 1 hours, shaking at 200-300 rpm
Pipette 100 µL of each transformation onto petri plates
Spin down cells at 6800 g for 3 min and discard 800 µL of the supernatant. Resuspend the cells in the remaining 100 µL, and pipette each transformation onto petri plates
Incubate transformations overnight (14-18 hr) at 37°C
Pick single colonies for PCR
Count colonies for control transformation

Cell Measurement

Pick 2 colonies from each of the transformation plates and inoculate in 5-10 mL LB medium + Chloramphenicol. Grow the cells overnight (16-18 hours) at 37°C and 220 rpm
Make a 1:10 dilution of each overnight culture in LB + Chloramphenicol (0.5 mL of culture into 4.5 mL of LB + Chlor)
Measure Abs₆₀₀ of these 1:10 diluted cultures
Record the data in your notebook
Dilute the cultures further to a target Abs₆₀₀ of 0.02 in a final volume of 12 mL LB medium + Chloramphenicol in 50 mL falcon tube (amber, or covered with foil to block light)
Take 500 µL samples of the diluted cultures at 0 hours into 1.5 mL Eppendorf tubes, prior to incubation. (At each time point 0 hours and 6 hours, you will take a sample from each of the 8 devices, two colonies per device, for a total of 16 Eppendorf tubes with 500 µL samples per time point, 32 samples total). Place the samples on ice
Incubate the remainder of the cultures at 37°C and 220 rpm for 6 hours
Take 500 µL samples of the cultures at 6 hours of incubation into 1.5 mL Eppendorf tubes. Place samples on ice
At the end of sampling point you need to measure your samples (Abs₆₀₀ and fluorescence measurement), see the below for details
Record data in your notebook
Import data into Excel sheet provided (fluorescence measurement tab)

CFU per 0.1 OD₆₀₀ E.coli Cultures

Culture colonies for two Positive Control (BBa_I20270) cultures and your two Negative Control (BBa_R0040) cultures for 16-18 hours
Dilute the overnight culture to OD₆₀₀ = 0.1 in 1 mL of LB + Cam media. Do this in triplicate for each culture. Check the OD₆₀₀ and make sure it is 0.1
Aseptically spead plate 100 µL on LB + Cam plates for those Final Dilution Factor is 8 x 10⁴ or 8 x 10⁵ or 8 x 10⁶
Incubate at 37°C overnight and count colonies after 18-20 hours of growth
Count the colonies on each plate with fewer than 300 colonies. Multiple the colony count by the Final Dilution Factor on each plate

Results

OD₆₀₀ Reference Point

According to the Reference OD₆₀₀, We calculate the final result:
OD₆₀₀/Abs₆₀₀=3.818
All cell density readings using this instrument with the same settings and volume can be converted to OD₆₀₀, so that we can use this ratio to convert subsequent experimental data.

Particle Standard Curve

We prepare a dilution series of monodisperse silica microspheres and measure the Abs₆₀₀ in plate reader. The size and optical characteristics of these microspheres are similar to cells, and there is a known amount of particles per volume. This measurement allows us to construct a standard curve of particle concentration which can be used to convert Abs₆₀₀ measurements to an estimated number of cells.

The final result:
mean of med-high levels=6.24E+08

Fluorescein Standard Curve

We prepare a dilution series of fluorescein in four replicates and measure the fluorescence in a 96 well plate in plate reader. By measuring these we generate a standard curve of fluorescence for fluorescein concentration. We will be able to use this to convert our cell based readings to an equivalent fluorescein concentration.

Final results:
Mean µM fluorescein / a.u.=3.60E-05
MEFL / a.u.=2.17E+08

Raw Plate Reader Measurements

Top

@@ Line 3: / Line 3: @@
 <head>
 <style type="text/css">
-      * { /*设置全局属性*/
-    position: relative;
-    -moz-box-sizing: border-box;
-         box-sizing: border-box;
-    margin: 0; padding: 0;
-    list-style: none;
-}
-}
 .subnavpicture .img2{display: none;}
 .subnavpicture:hover .img1{display: none;}
 .subnavpicture:hover .img2{display: block;}
-.component {
+.subnavpicture {
-    height: 13em; /*扇形区域的高度(半径)*/
+      position: absolute;   /*使其绝对定位到框中央(圆心位置)，便于旋转*/
-    margin-top: 22%;
-    margin-left: -100px;
-    position: fixed;
-    z-index: 1000;
-}
-.component button:focus {
-	outline: none;
-}/*隐藏按钮/链接点击后出现的虚线方框代码*/
-.component button::-moz-focus-inner {
-	border-color: transparent !important;
-}/*mozilla*/
-.component button {
-    position: absolute;   /*使其绝对定位到框中央(圆心位置)，便于旋转*/
      top: 10%; left: 5%;
      margin-top: -2.25em;  /*抵消绝对定位，使其仍然从左上角开始显示*/
@@ Line 45: / Line 23: @@
      -webkit-backface-visibility: hidden; /*当元素不面向屏幕时(背面)不可见*/
 }
-.component button:hover {
+.component {
-     font-weight: 750;
+     height: 13em; /*扇形区域的高度(半径)*/
+    top: 50%;
+    left: 1.8%;
+    position:fixed;
+    z-index: 100;
 }
 .round-nav {
-    position: absolute;   /*绝对定位到导航框中央*/
-    top: 10%; left: 5%; /*使导航元素的起始位置在圆心处*/
-    width: 27em; height: 27em;
-    margin-top: -13em;    /*抵消绝对定位，使导航仍然从左上角开始显示*/
-    margin-left: -13.5em; /*目的是使圆心归到正中央，但不影响显示*/
-    z-index: 10; /*比Button第一层，被覆盖*/
-    /*border-radius: 50%;*/
-    background: transparent;
-    opacity: 0;
-    -webkit-transition: all 0.3s ease 0.2s; /*设定动画过渡样式和时长*/
-       -moz-transition: all 0.3s ease 0.2s;
-            transition: all 0.3s ease 0.2s;
-    -webkit-transform: scale(0.1); /*设定动画效果(缩小)*/
-       -moz-transform: scale(0.1);
-        -ms-transform: scale(0.1);
-            transform: scale(0.1);
-    pointer-events: none; /*忽略该区域的鼠标事件，使其不遮挡下方物体的鼠标效果*/
-    overflow: hidden;
-}
-/*覆盖锚链接后的空白，防止其可点击*/
-.round-nav:after{ /*实际上可以去掉这个效果*/
-  content:".";
-  display:block;
-  font-size:2em;
-  width:6.2em;
-  height:6.2em;
    position: absolute;
-   left: 50%;
+   z-index: 150;
-  margin-left: -3.1em;
+   display: none;
-   top:50%;
+   top: -65%;
-   margin-top: -3.1em;
+   left: 10px;
-  border-radius: 50%;
-   z-index:10;
-  color: transparent; /*透明，不显示*/
 }
-.round-nav li {
+.opened-nav {
-    position: absolute; /*定位到中央，原理同上*/
+  position:absolute;
-    top: 50%; left: 50%;
+  top: -65%;
-    width: 10em; height: 10em;
+  left: 10px;
-    margin-top: -1.3em;
+  z-index: 150;
-    margin-left: -10em;
+  display: block;
-    overflow: hidden; /*遮挡其他区域，显示出扇形*/
-    font-size: 1.5em;
-    -webkit-transition: all .3s ease;
-       -moz-transition: all .3s ease;
-            transition: all .3s ease;
-    -webkit-transform: rotate(76deg) skew(60deg); /*skew(60deg)表示元素翻转60度*/
-       -moz-transform: rotate(76deg) skew(60deg); /*翻转之后呈现平行四边形*/
-        -ms-transform: rotate(76deg) skew(60deg); /*旋转76度，是为了使所有元素都先从上方出现*/
-            transform: rotate(76deg) skew(60deg);
-    -webkit-transform-origin: 100% 100%;
-       -moz-transform-origin: 100% 100%;
-            transform-origin: 100% 100%;
-    pointer-events: none;
 }
+#one1 {
-/*TUDO：在列表上使用TAB键会出现奇怪问题*/
+  margin-bottom: 10px;
-.round-nav li a {
-    position: absolute;
-    position: fixed; /*a跟随li运动*/
-    right: -7.25em;
-    bottom: -7.25em;
-    width: 14.5em;
-    height: 14.5em;
-    display: block;
-    border-radius: 50%;
-    color: white;
-    text-align: center;
-    text-decoration: none;
-    font-size: 1.2em;
-    line-height: 2;
-    background: rgb(3,23,23);
-    background: -webkit-radial-gradient(transparent 35%, rgb(3,23,23) 35%);
-    background:    -moz-radial-gradient(transparent 35%, rgb(3,23,23) 35%);
-    background:         radial-gradient(transparent 35%, rgb(3,23,23) 35%);
-    -webkit-transform: skew(-60deg) rotate(-75deg) scale(1); /*旋转和翻转是为了和li保持相对一致*/
-       -moz-transform: skew(-60deg) rotate(-75deg) scale(1);
-        -ms-transform: skew(-60deg) rotate(-75deg) scale(1);
-            transform: skew(-60deg) rotate(-75deg) scale(1);
-    -webkit-backface-visibility: hidden;
-            backface-visibility: hidden;
-    pointer-events: auto;
 }
-.round-nav li a:hover,
+#two2 {
-.round-nav li a:active,  /*从透明到线绿色径向渐变*/
+  margin-bottom: 10px;
-.round-nav li a:focus {
-	color: white;
-    background: -webkit-radial-gradient(transparent 35%, rgba(3,23,23,.70) 35%);
-    background:    -moz-radial-gradient(transparent 35%, rgba(3,23,23,.70) 35%);
-    background:         radial-gradient(transparent 35%, rgba(3,23,23,.70) 35%);
-}
-.round-nav li a span {
-    position: relative; /*跟随a运动*/
-    top: 1.8em;
-    display: block;
-    font-size: 0.5em;
-    font-weight: 600;
-    text-transform: uppercase;
 }
-.opened-nav {  /*设定导航打开后的效果，对应.round-nav*/
-    border-radius: 50%;
-    opacity: 1;
-    -webkit-transition: all .3s ease;
-       -moz-transition: all .3s ease;
-            transition: all .3s ease;
-    -webkit-transform: scale(1); /*先将导航放大*/
-       -moz-transform: scale(1); /*表现为：点击后，从按钮背后伸展到正上方*/
-        -ms-transform: scale(1);
-            transform: scale(1);
-    pointer-events: auto;
-}
-.opened-nav li {
-    -webkit-transition: all .3s ease .15s;
-       -moz-transition: all .3s ease .15s;
-            transition: all .3s ease .15s;
-}
-/*使不同的li元素旋转不同的角度*/
-.opened-nav li:nth-child(1) {
-    -webkit-transform: rotate(136deg) skew(60deg);
-       -moz-transform: rotate(136deg) skew(60deg);
-        -ms-transform: rotate(136deg) skew(60deg);
-            transform: rotate(136deg) skew(60deg);
-}
-.opened-nav li:nth-child(2) {
-    -webkit-transform: rotate(168deg) skew(60deg);
-       -moz-transform: rotate(168deg) skew(60deg);
-        -ms-transform: rotate(168deg) skew(60deg);
-            transform: rotate(168deg) skew(60deg);
-}
-.opened-nav li:nth-child(3) {
-    -webkit-transform: rotate(200deg) skew(60deg);
-       -moz-transform: rotate(200deg) skew(60deg);
-        -ms-transform: rotate(200deg) skew(60deg);
-            transform: rotate(200deg) skew(60deg);
-}
 .panelheadingcursor {
    cursor: pointer;
@@ Line 199: / Line 59: @@
              function toggleClass(){
                  wrapper = document.getElementById('nav-wrapper');
-                open = !open;
+                     wrapper.className = "opened-nav";
-                if (open === true) {
-                     wrapper.className = "round-nav opened-nav";
-                } else {
-                    wrapper.className = "round-nav";
-                }
              }
              function toggledispear(){
@@ Line 212: / Line 67: @@
      </script>
-    <!-- HTML5 shim and Respond.js for IE8 support of HTML5 elements and media queries -->
-    <!-- WARNING: Respond.js doesn't work if you view the page via file:// -->
-    <!--[if lt IE 9]>
-      <script src="https://oss.maxcdn.com/html5shiv/3.7.3/html5shiv.min.js"></script>
-      <script src="https://oss.maxcdn.com/respond/1.4.2/respond.min.js"></script>
-    <![endif]-->
 </head>
 <body>
      <div>
-       <img class="myBackground" src="images/background.jpg">
+       <img class="myBackground" src="https://static.igem.org/mediawiki/2018/6/64/T--NKU_CHINA--background.jpg">
      </div>
        <div class="container-fluid">
        	<div class="row">
          <div class="component col-xs-4 col-xs-push-1">
-            <button onmouseover="toggleClass()">
+              <div class="subnavpicture" onmouseover="toggleClass()">
-              <div class="subnavpicture">
+                 <img class="img1" src="https://static.igem.org/mediawiki/2018/3/32/T--NKU_CHINA--cebian1.png" style="height: 250px;width: auto;">
-                 <img class="img1" src="images/cebian1.png" style="height: 250px;width: auto;">
+                 <img class="img2" src="https://static.igem.org/mediawiki/2018/7/7e/T--NKU_CHINA--cebian2.png" style="height: 250px;width: auto;">
-                 <img class="img2" src="images/cebian2.png" style="height: 250px;width: auto;">
                </div>
-            </button>
              <div class="round-nav" id="nav-wrapper" onclick="toggledispear()">
-                 <ul>
+                 <ul class="list-unstyled">
-                     <li><a href="#firstpart" data-toogle="tooltip" data-placement="right" title="Materials"><span><img src="images/materials.png" style="width: 50px;height: auto;"></span></a></li>
+                     <li id="one1"><a href="#firstpart" data-toogle="tooltip" data-placement="right" title="Materials"><span><img src="https://static.igem.org/mediawiki/2018/6/63/T--NKU_CHINA--interlab_subnav1.png" style="width: 50px;height: auto;"></span></a></li>
-                     <li><a href="#secondpart" data-toogle="tooltip" data-placement="right" title="Methods"><span><img src="images/methods.png" style="width: 50px;height: auto;"></span></a></li>
+                     <li id="two2"><a href="#secondpart" data-toogle="tooltip" data-placement="right" title="Methods"><span><img src="https://static.igem.org/mediawiki/2018/a/a6/T--NKU_CHINA--interlab_subnav2.png" style="width: 50px;height: auto;"></span></a></li>
-                     <li><a href="#thirdpart" data-toogle="tooltip" data-placement="right" title="Results"><span><img src="images/results.png" style="width: 40px;height: auto;"></span></a></li>
+                     <li id="three3"><a href="#thirdpart" data-toogle="tooltip" data-placement="right" title="Results"><span><img src="https://static.igem.org/mediawiki/2018/a/ac/T--NKU_CHINA--interlab_subnav3.png" style="width: 40px;height: auto;"></span></a></li>
                   </ul>
              </div>
@@ Line 243: / Line 89: @@
      </div>
 </div>
      <main>
@@ Line 255: / Line 102: @@
    <div class="panel panel-default" style="margin-right: 10%;margin-left: 10%;">
      <div class="panel-heading panelheadingcursor" data-toggle="collapse" data-parent="#materials" data-target="#collapse1" style="background-color: #B5CACF;">
-       <h4 class="panel-title" style="height: 30px;">
+       <h4 class="panel-title" style="height: 35px;">
          <a style="font-size: 25px;">Reagents and Apparatus</a>
        </h4>
@@ Line 264: / Line 111: @@
 <li style="font-size: 20px;line-height: 25px;">Competent cells (<i>Escherichia coli</i> strain DH5&#945;)</li>
 <li style="font-size: 20px;line-height: 25px;">1 mL LUDOX CL-X (provided in kit) </li>
-<li style="font-size: 20px;line-height: 25px;">300 &#181L Silica beads - Microsphere suspension (provided in kit, 4.7 x 10<sup>8</sup> microspheres)</li>
+<li style="font-size: 20px;line-height: 25px;">300 &#181;L Silica beads - Microsphere suspension (provided in kit, 4.7 x 10<sup>8</sup> microspheres)</li>
 <li style="font-size: 20px;line-height: 25px;">LB (Luria Bertani) media</li>
 <li style="font-size: 20px;line-height: 25px;">Fluorescein (provided in kit) </li>
@@ Line 282: / Line 129: @@
 <div class="panel panel-default" style="margin-right: 10%;margin-left: 10%;">
      <div class="panel-heading panelheadingcursor" data-toggle="collapse" data-parent="#materials" data-target="#collapse2" style="background-color: #D5E1E4;">
-       <h4 class="panel-title" style="height: 30px;" id="secondpart">
+       <h4 class="panel-title" style="height: 35px;" id="secondpart">
          <a style="font-size: 25px;">Devices</a>
        </h4>
@@ Line 309: / Line 156: @@
    <div class="panel panel-default" style="margin-right: 10%;margin-left: 10%;">
      <div class="panel-heading panelheadingcursor" data-toggle="collapse" data-parent="#methods" data-target="#collapse3" style="background-color: #89B777;">
-       <h4 class="panel-title" style="height: 30px;">
+       <h4 class="panel-title" style="height: 35px;">
          <a style="font-size: 25px;">OD<sub>600</sub> Reference Point</a>
        </h4>
@@ Line 316: / Line 163: @@
        <div class="panel-body">
          <ul class="interlab-list">
-           <li style="font-size: 20px;line-height: 25px;">Add 100 &#181L LUDOX into wells A1, B1, C1, D1</li>
+           <li style="font-size: 20px;line-height: 25px;">Add 100 &#181;L LUDOX into wells A1, B1, C1, D1</li>
-             <li style="font-size: 20px;line-height: 25px;">Add 100 &#181L of dd H<sub>2</sub>O into wells A2, B2, C2, D2</li>
+             <li style="font-size: 20px;line-height: 25px;">Add 100 &#181;L of dd H<sub>2</sub>O into wells A2, B2, C2, D2</li>
              <li style="font-size: 20px;line-height: 25px;">Measure absorbance at 600 nm of all samples in the measurement mode you plan to use for cell measurements</li>
              <li style="font-size: 20px;line-height: 25px;">Record the data in the table below or in your notebook</li>
@@ Line 328: / Line 175: @@
   <div class="panel panel-default" style="margin-right: 10%;margin-left: 10%;">
      <div class="panel-heading panelheadingcursor" data-toggle="collapse" data-parent="#methods" data-target="#collapse4" style="background-color: #95BF85;">
-       <h4 class="panel-title" style="height: 30px;">
+       <h4 class="panel-title" style="height: 35px;">
          <a style="font-size: 25px;">Particle Standard Curve</a>
        </h4>
@@ Line 336: / Line 183: @@
          <ul class="interlab-list">
            <li style="font-size: 20px;line-height: 25px;">Obtain the tube labeled "Silica Beads" from the InterLab test kit and vortex vigorously for 30 seconds</li>
-             <li style="font-size: 20px;line-height: 25px;">Immediately pipet 96 &#181L microspheres into a 1.5 mL eppendorf tube</li>
+             <li style="font-size: 20px;line-height: 25px;">Immediately pipet 96 &#181;L microspheres into a 1.5 mL eppendorf tube</li>
-             <li style="font-size: 20px;line-height: 25px;">Add 904 &#181L of ddH<sub>2</sub>O to the microspheres</li>
+             <li style="font-size: 20px;line-height: 25px;">Add 904 &#181;L of ddH<sub>2</sub>O to the microspheres</li>
              <li style="font-size: 20px;line-height: 25px;">Vortex well. This is your Microsphere Stock Solution</li>
              <li style="font-size: 20px;line-height: 25px;">Repeat dilution series for rows B, C, D</li>
@@ Line 351: / Line 198: @@
     <div class="panel panel-default" style="margin-right: 10%;margin-left: 10%;">
      <div class="panel-heading panelheadingcursor" data-toggle="collapse" data-parent="#methods" data-target="#collapse5" style="background-color: #ADCEA1;">
-       <h4 class="panel-title" style="height: 30px;">
+       <h4 class="panel-title" style="height: 35px;">
          <a style="font-size: 25px;">Fluorescence Standard Curve</a>
        </h4>
@@ Line 359: / Line 206: @@
          <ul class="interlab-list">
            <li style="font-size: 20px;line-height: 25px;">Spin down fluorescein kit tube to make sure pellet is at the bottom of tube. </li>
-  <li style="font-size: 20px;line-height: 25px;">Prepare 10x fluorescein stock solution (100 &#181M) by resuspending fluorescein in 1 mL of 1xPBS</li>
+  <li style="font-size: 20px;line-height: 25px;">Prepare 10x fluorescein stock solution (100 &#181;M) by resuspending fluorescein in 1 mL of 1xPBS</li>
-<li style="font-size: 20px;line-height: 25px;">Dilute the 10x fluorescein stock solution with 1xPBS to make a 1x fluorescein solution with concentration 10 &#181M: 100 &#181L of 10x fluorescein stock into 900 &#181L 1xPBS</li>
+<li style="font-size: 20px;line-height: 25px;">Dilute the 10x fluorescein stock solution with 1xPBS to make a 1x fluorescein solution with concentration 10 &#181;M: 100 &#181;L of 10x fluorescein stock into 900 &#181;L 1xPBS</li>
-<li style="font-size: 20px;line-height: 25px;">Add 100 &#181L of PBS into wells A2, B2, C2, D2...A12, B12, C12, D12</li>
+<li style="font-size: 20px;line-height: 25px;">Add 100 &#181;L of PBS into wells A2, B2, C2, D2...A12, B12, C12, D12</li>
-<li style="font-size: 20px;line-height: 25px;">Add 200 &#181L of fluorescein 1x stock solution into A1, B1, C1, D1</li>
+<li style="font-size: 20px;line-height: 25px;">Add 200 &#181;L of fluorescein 1x stock solution into A1, B1, C1, D1</li>
-<li style="font-size: 20px;line-height: 25px;">Transfer 100 &#181L of fluorescein stock solution from A1 into A2</li>
+<li style="font-size: 20px;line-height: 25px;">Transfer 100 &#181;L of fluorescein stock solution from A1 into A2</li>
-<li style="font-size: 20px;line-height: 25px;">Mix A2 by pipetting up and down 3x and transfer 100 &#181L into A3...</li>
+<li style="font-size: 20px;line-height: 25px;">Mix A2 by pipetting up and down 3x and transfer 100 &#181;L into A3...</li>
-<li style="font-size: 20px;line-height: 25px;">Mix A3 by pipetting up and down 3x and transfer 100 &#181L into A4...</li>
+<li style="font-size: 20px;line-height: 25px;">Mix A3 by pipetting up and down 3x and transfer 100 &#181;L into A4...</li>
-<li style="font-size: 20px;line-height: 25px;">Mix A4 by pipetting up and down 3x and transfer 100 &#181L into A5...</li>
+<li style="font-size: 20px;line-height: 25px;">Mix A4 by pipetting up and down 3x and transfer 100 &#181;L into A5...</li>
-<li style="font-size: 20px;line-height: 25px;">Mix A5 by pipetting up and down 3x and transfer 100 &#181L into A6...</li>
+<li style="font-size: 20px;line-height: 25px;">Mix A5 by pipetting up and down 3x and transfer 100 &#181;L into A6...</li>
-<li style="font-size: 20px;line-height: 25px;">Mix A6 by pipetting up and down 3x and transfer 100 &#181L into A7...</li>
+<li style="font-size: 20px;line-height: 25px;">Mix A6 by pipetting up and down 3x and transfer 100 &#181;L into A7...</li>
-<li style="font-size: 20px;line-height: 25px;" style="font-size: 20px;line-height: 25px;">Mix A7 by pipetting up and down 3x and transfer 100 &#181L into A8...</li>
+<li style="font-size: 20px;line-height: 25px;" style="font-size: 20px;line-height: 25px;">Mix A7 by pipetting up and down 3x and transfer 100 &#181;L into A8...</li>
-<li style="font-size: 20px;line-height: 25px;">Mix A8 by pipetting up and down 3x and transfer 100 &#181L into A9...</li>
+<li style="font-size: 20px;line-height: 25px;">Mix A8 by pipetting up and down 3x and transfer 100 &#181;L into A9...</li>
-<li style="font-size: 20px;line-height: 25px;">Mix A9 by pipetting up and down 3x and transfer 100 &#181L into A10...</li>
+<li style="font-size: 20px;line-height: 25px;">Mix A9 by pipetting up and down 3x and transfer 100 &#181;L into A10...</li>
-<li style="font-size: 20px;line-height: 25px;">Mix A10 by pipetting up and down 3x and transfer 100 &#181L into A11...</li>
+<li style="font-size: 20px;line-height: 25px;">Mix A10 by pipetting up and down 3x and transfer 100 &#181;L into A11...</li>
-<li style="font-size: 20px;line-height: 25px;">Mix A11 by pipetting up and down 3x and transfer 100 &#181L into liquid waste</li>
+<li style="font-size: 20px;line-height: 25px;">Mix A11 by pipetting up and down 3x and transfer 100 &#181;L into liquid waste</li>
 <li style="font-size: 20px;line-height: 25px;">Repeat dilution series for rows B, C, D</li>
 <li style="font-size: 20px;line-height: 25px;">Measure fluorescence of all samples in instrument</li>
@@ Line 385: / Line 232: @@
 <div class="panel panel-default" style="margin-right: 10%;margin-left: 10%;">
      <div class="panel-heading panelheadingcursor" data-toggle="collapse" data-parent="#methods" data-target="#collapse6" style="background-color: #C3DBBA;">
-       <h4 class="panel-title" style="height: 30px;">
+       <h4 class="panel-title" style="height: 35px;">
          <a style="font-size: 25px;">Competent Cells and Transformation</a>
        </h4>
@@ Line 394: / Line 241: @@
            <li style="font-size: 20px;line-height: 25px;">Resuspend DNA in selected wells in the Distribution Kit with 10 &#181L dH<sub>2</sub>O</li>
 <li style="font-size: 20px;line-height: 25px;">Thaw competent cells on ice</li>
-<li style="font-size: 20px;line-height: 25px;">Pipette 50 &#181L of competent cells into 1.5 mL tube</li>
+<li style="font-size: 20px;line-height: 25px;">Pipette 50 &#181;L of competent cells into 1.5 mL tube</li>
-<li style="font-size: 20px;line-height: 25px;">Pipette 1 &#181L of resuspended DNA into 1.5 mL tube</li>
+<li style="font-size: 20px;line-height: 25px;">Pipette 1 &#181;L of resuspended DNA into 1.5 mL tube</li>
-<li style="font-size: 20px;line-height: 25px;">Pipette 1 &#181L of control DNA into 2 mL tube</li>
+<li style="font-size: 20px;line-height: 25px;">Pipette 1 &#181;L of control DNA into 2 mL tube</li>
 <li style="font-size: 20px;line-height: 25px;">Close 1.5 mL tubes, incubate on ice for 30 min</li>
 <li style="font-size: 20px;line-height: 25px;">Heat shock tubes at 42&#176;C for 45 sec</li>
 <li style="font-size: 20px;line-height: 25px;">Incubate on ice for 5 min</li>
-<li style="font-size: 20px;line-height: 25px;">Pipette 950 &#181L SOC media to each transformation</li>
+<li style="font-size: 20px;line-height: 25px;">Pipette 950 &#181;L SOC media to each transformation</li>
 <li style="font-size: 20px;line-height: 25px;">Incubate at 37&#176;C for 1 hours, shaking at 200-300 rpm</li>
-<li style="font-size: 20px;line-height: 25px;">Pipette 100 &#181L of each transformation onto petri plates</li>
+<li style="font-size: 20px;line-height: 25px;">Pipette 100 &#181;L of each transformation onto petri plates</li>
-<li style="font-size: 20px;line-height: 25px;">Spin down cells at 6800 g for 3 min and discard 800 &#181L of the supernatant. Resuspend the cells in the remaining 100 &#181L, and pipette each transformation onto petri plates</li>
+<li style="font-size: 20px;line-height: 25px;">Spin down cells at 6800 g for 3 min and discard 800 &#181;L of the supernatant. Resuspend the cells in the remaining 100 &#181;L, and pipette each transformation onto petri plates</li>
 <li style="font-size: 20px;line-height: 25px;">Incubate transformations overnight (14-18 hr) at 37&#176;C</li>
 <li style="font-size: 20px;line-height: 25px;">Pick single colonies for PCR</li>
@@ Line 409: / Line 256: @@
 </ul>
        </div>
-     </div> style="font-size: 20px;line-height: 25px;"
+     </div>
    </div>
 <div class="panel panel-default" style="margin-right: 10%;margin-left: 10%;">
      <div class="panel-heading panelheadingcursor" data-toggle="collapse" data-parent="#methods" data-target="#collapse7" style="background-color: #D3E5CE;">
-       <h4 class="panel-title" style="height: 30px;">
+       <h4 class="panel-title" style="height: 35px;">
          <a style="font-size: 25px;">Cell Measurement</a>
        </h4>
@@ Line 426: / Line 273: @@
 <li style="font-size: 20px;line-height: 25px;">Record the data in your notebook</li>
 <li style="font-size: 20px;line-height: 25px;">Dilute the cultures further to a target Abs<sub>600</sub> of 0.02 in a final volume of 12 mL LB medium + Chloramphenicol in 50 mL falcon tube (amber, or covered with foil to block light)</li>
-<li style="font-size: 20px;line-height: 25px;">Take 500 &#181L samples of the diluted cultures at 0 hours into 1.5 mL Eppendorf tubes, prior to incubation. (At each time point 0 hours and 6 hours, you will take a sample from each of the 8 devices, two colonies per device, for a total of 16 Eppendorf tubes with 500 &#181L samples per time point, 32 samples total). Place the samples on ice</li>
+<li style="font-size: 20px;line-height: 25px;">Take 500 &#181;L samples of the diluted cultures at 0 hours into 1.5 mL Eppendorf tubes, prior to incubation. (At each time point 0 hours and 6 hours, you will take a sample from each of the 8 devices, two colonies per device, for a total of 16 Eppendorf tubes with 500 &#181;L samples per time point, 32 samples total). Place the samples on ice</li>
 <li style="font-size: 20px;line-height: 25px;">Incubate the remainder of the cultures at 37&#176;C and 220 rpm for 6 hours</li>
-<li style="font-size: 20px;line-height: 25px;">Take 500 &#181L samples of the cultures at 6 hours of incubation into 1.5 mL Eppendorf tubes. Place samples on ice</li>
+<li style="font-size: 20px;line-height: 25px;">Take 500 &#181;L samples of the cultures at 6 hours of incubation into 1.5 mL Eppendorf tubes. Place samples on ice</li>
 <li style="font-size: 20px;line-height: 25px;">At the end of sampling point you need to measure your samples (Abs<sub>600</sub> and fluorescence measurement), see the below for details</li>
 <li style="font-size: 20px;line-height: 25px;">Record data in your notebook</li>
@@ Line 436: / Line 283: @@
      </div>
    </div>
- style="font-size: 20px;line-height: 25px;"
    <div class="panel panel-default" style="margin-right: 10%;margin-left: 10%;">
      <div class="panel-heading panelheadingcursor" data-toggle="collapse" data-parent="#methods" data-target="#collapse8" style="background-color: #E7F1E4;">
-       <h4 class="panel-title" style="height: 30px;"  id="thirdpart">
+       <h4 class="panel-title" style="height: 35px;"  id="thirdpart">
          <a style="font-size: 25px;">CFU per 0.1 OD<sub>600</sub> <i>E.coli</i> Cultures</a>
        </h4>
@@ Line 448: / Line 295: @@
            <li style="font-size: 20px;line-height: 25px;">Culture colonies for two Positive Control (BBa_I20270) cultures and your two Negative Control (BBa_R0040) cultures for 16-18 hours</li>
 <li style="font-size: 20px;line-height: 25px;">Dilute the overnight culture to OD<sub>600</sub> = 0.1 in 1 mL of LB + Cam media. Do this in triplicate for each culture. Check the OD<sub>600</sub> and make sure it is 0.1</li>
-<li style="font-size: 20px;line-height: 25px;">Aseptically spead plate 100 &#181L on LB + Cam plates for those Final Dilution Factor is 8 x 10<sup>4</sup> or 8 x 10<sup>5</sup> or 8 x 10<sup>6</sup></li>
+<li style="font-size: 20px;line-height: 25px;">Aseptically spead plate 100 &#181;L on LB + Cam plates for those Final Dilution Factor is 8 x 10<sup>4</sup> or 8 x 10<sup>5</sup> or 8 x 10<sup>6</sup></li>
 <li style="font-size: 20px;line-height: 25px;">Incubate at 37&#176;C overnight and count colonies after 18-20 hours of growth</li>
 <li style="font-size: 20px;line-height: 25px;">Count the colonies on each plate with fewer than 300 colonies. Multiple the colony count by the Final Dilution Factor on each plate</li>
@@ Line 462: / Line 309: @@
     <div class="panel panel-default" style="margin-right: 10%;margin-left: 10%;">
      <div class="panel-heading panelheadingcursor" data-toggle="collapse" data-parent="#results" data-target="#collapse9" style="background-color: #A098B6;">
-       <h4 class="panel-title" style="height: 30px;">
+       <h4 class="panel-title" style="height: 35px;">
          <a style="font-size: 25px;">OD<sub>600</sub> Reference Point</a>
        </h4>
@@ Line 479: / Line 326: @@
   <div class="panel panel-default" style="margin-right: 10%;margin-left: 10%;">
      <div class="panel-heading panelheadingcursor" data-toggle="collapse" data-parent="#results" data-target="#collapse10" style="background-color: #B7B1C8;">
-       <h4 class="panel-title" style="height: 30px;">
+       <h4 class="panel-title" style="height: 35px;">
          <a style="font-size: 25px;">Particle Standard Curve</a>
        </h4>
@@ Line 496: / Line 343: @@
 <div class="panel panel-default" style="margin-right: 10%;margin-left: 10%;">
      <div class="panel-heading panelheadingcursor"  data-toggle="collapse" data-parent="#results" data-target="#collapse11" style="background-color: #D1CDDC;">
-       <h4 class="panel-title" style="height: 30px;">
+       <h4 class="panel-title" style="height: 35px;">
          <a style="font-size: 25px;">Fluorescein Standard Curve</a>
        </h4>
@@ Line 506: / Line 353: @@
          <img src="https://static.igem.org/mediawiki/2018/d/d1/T--NKU_CHINA--FluoresceinStandardcurve_list.png" class="img-responsive center-block">
          <img src="https://static.igem.org/mediawiki/2018/0/00/T--NKU_CHINA--FluoresceinStandardCurve_logscale.png" class="img-responsive center-block">
-         <p style="font-size: 20px;line-height: 25px;margin-left: 10px;color: #000"> Final results:<br>Mean &#181M fluorescein / a.u.=3.60E-05<br>MEFL / a.u.=2.17E+08</p>
+         <p style="font-size: 20px;line-height: 25px;margin-left: 10px;color: #000"> Final results:<br>Mean &#181;M fluorescein / a.u.=3.60E-05<br>MEFL / a.u.=2.17E+08</p>
   </div>
      </div>
@@ Line 513: / Line 360: @@
 <div class="panel panel-default" style="margin-right: 10%;margin-left: 10%;">
      <div class="panel-heading panelheadingcursor" data-toggle="collapse" data-parent="#results" data-target="#collapse12" style="background-color: #EAE8EF;">
-       <h4 class="panel-title" style="height: 30px;">
+       <h4 class="panel-title" style="height: 35px;">
          <a style="font-size: 25px;">Raw Plate Reader Measurements</a>
        </h4>
@@ Line 528: / Line 375: @@
 </div>
 </main>
+<footer>
+	<div class="container-fluid myFooter">
+		<div class="row">
+			<div class="col-xs-8 col-xs-push-1">
+				<img src="https://static.igem.org/mediawiki/2018/0/0b/T--NKU_CHINA--duihui_final.png" style="height: 250px; width: auto;">
+			</div>
+			<div class="col-xs-4 footer-w3ls">
+				<h3 style="color: white; font-size: 30px;">Contacts</h3>
+				<ul class="social-icons1" style="margin:0 auto;padding-top:0;">
+				<li><a href="#"><i class="fa fa-facebook" aria-hidden="true"></i></a></li>
+				<li><a href="https://twitter.com/iGEMNKU2018"><i class="fa fa-twitter" aria-hidden="true"></i></a></li>
+				<li><a href="mailto:nkuigem2018@163.com"><i class="fa fa fa-envelope-o" aria-hidden="true"></i></a></li>
+			    </ul>
+			    <h3 style="color: white; font-size: 30px;">Address</h3>
+                <address style="color: white; font-size: 17px;">
+                    Nankai University</br>
+                    No.94 Weijin Road, Nankai District</br>
+                    Tianjin, P.R.China 300071</br>
+                </address>
+            </div>
+            <div class="col-xs-12">
+            	<p class="Copyright" style="text-align: center; color: white;">Copyright © Team NKU_CHINA 2018</p>
+        </div>
+    </div>
+</footer>
 <a href="#0" class="cd-top">Top</a>
 </body>
 </html>
-{{Template:NKU_CHINA/footer}}