Revision as of 12:00, 1 November 2017

<body>

       <a href="javascript:void(0)" class="closebtn" onclick="closeNav()">×</a>
       <a href="#abstract">Abstract</a>
       <a href="#plasmids">Plasmids and the prediction of results</a>
       <a href="#component">Component cell strain & 96 well plate & Plate reader</a>
       <a href="#protocol">Protocol</a>
       <a href="#calibrationod600">Calibration —— OD600 reference point</a>
       <a href="#calibrationflorescein">Calibration —— fluorescein fluorescence standard curve

Materials</a>

       <a href="#cellmeasurement">Cell measurement</a>
       <a href="#cellmeasurement2">Cell measurement</a>
       <a href="#results">Final Results</a>

Repeatability is one of the most important part of biological experiment, however, it’s difficult to repeat the measurement data in different labs. As for the measurement of fluorescence, it can be influenced by various factors: bacterial strain, culture medium, plate reader and so on. Therefore, following a standard protocol strictly is one of the guarantee of the repeatable experimental conclusion. This year, we use plate reader rather than flow cytometry in 2016 (<a href="https://2016.igem.org/Team:ShanghaitechChina/InterLab">click here to view</a>). Except for individual settings, we follow the standard protocol by iGEM authority completely, the data is reliable.</br>

Plasmids and the prediction of results

All the eight plasmids are shown below:

               

               <img align="middle" src="" width="800" height="400" alt=""></img>
               

               

               <p align="left" style="text-indent: 21pt;">From the table above, there are three different constitutive promotors (pcons) and two different ribosome binding sites (RBSs), which form six different combinations as Test Device 1-8. </p>

                   <img align="middle" src="" width="250" height="400" alt="">

                   <a href="http://parts.igem.org/Part:BBa_J23101">(http://parts.igem.org/Part:BBa_J23101)</a></img>

               <p class="MsoNormal" align="left" style="text-indent: 21pt;">
                   According
                       to the data of iGEM parts, we know that the strength of these three pcons: J23101＞J23106＞
                   
                          J23117. However, there lacks the
                          reliable data of the strength of pcon (J23151) and the comparison of three RBSs
                          (B0032, B0034, J364100). In conclusion, we predict the result of the Interlab fluorescence
                          measurement: 
                          <o:p></o:p>
                       
               </p>
               <p class="MsoNormal" align="left">
                          <a name="OLE_LINK2"></a><a name="OLE_LINK1">Test Device 1 </a>＞ Test Device 2 ＞ Test Device 3
                   
                          <o:p></o:p>
                       
               </p>
               <p class="MsoNormal" align="left" style="text-indent: 21pt;">
                   Test Device 4 ＞ Test Device 5 ＞
                   
                          Test Device 6
                          <o:p></o:p>
                       
               </p>
               <p class="MsoNormal" align="left" style="text-indent: 21pt;">
                   
                          All the plasmid sequences (.dna file) can be download from: <a href="https://drive.google.com/file/d/0Bxb6U-RiSYsgQ1pTU0FsTG1SY1k/view?usp=sharing">https://drive.google.com/file/d/0Bxb6U-RiSYsgQ1pTU0FsTG1SY1k/view?usp=sharing</a>
                          <o:p></o:p>
                       
               </p>

Component cell strain & 96 well plate & Plate reader

           

           <p style="text-indent: 21pt;">
               Component cell strain

Brand: 康为世纪 (cwbiotech)

Strain: E.coli K-12 DH5α

96 well plate

Brand: Corning

-Black plate

-Flat-bottomed wells

Plate reader

Brand: BioTek

Instrument model: Cytation 5

Serial number: 1511021F

Pathlength correction: No

Number of flashes per well: 10

Orbital averaging (mm): 3.5

Fluorescence reading: top optic

Filter: Yes

Excitation wavelength (nm): 485/9

Emission wavelength (nm): 528/9

Protocol

(Some of the following steps are different from standard protocol from iGEM authority, the changes and the reasons are shown as red.) <o:p></o:p>

Calibration —— OD600 reference point <o:p></o:p>

Materials <o:p></o:p>

1ml LUDOX <o:p></o:p>

ddH₂O <o:p></o:p>

96 well plate <o:p></o:p>

Method <o:p></o:p>

◻ Add 100 µl LUDOX into wells A1, B1, C1, D1 <o:p></o:p>

◻ Add 100 µl of H₂ O into wells A2, B2, C2, D2 <o:p></o:p>

◻ Measure absorbance 600 nm of all samples in all standard measurement modes in instrument <o:p></o:p>

◻ Record the data in the table below <o:p></o:p>

◻ Import data into Excel (OD600 reference point tab ) Sheet_1 provided <o:p></o:p>

Calibration —— fluorescein fluorescence standard curve <o:p></o:p>

Materials <o:p></o:p>

fluorescein <o:p></o:p>

10mL 1X PBS <o:p></o:p>

96 well plate <o:p></o:p>

Method <o:p></o:p>

—— Prepare the fluorescein stock solution <o:p></o:p>

◻ Spin down fluorescein stock tube to make sure pellet is at the bottom of tube. <o:p></o:p>

(5000rpm, 5min) <o:p></o:p>

◻ Prepare 2X fluorescein stock solution (100 µM) by resuspending fluorescein in 1 mL of 1X PBS. <o:p></o:p>

◻ Dilute the 2X fluorescein stock solution with 1X PBS to make a 1X fluorescein solution and resulting concentration of fluorescein stock solution 50 µM. <o:p></o:p>

—— Prepare the serial dilutions of fluorescein <o:p></o:p>

◻ Add 100 µl of PBS into wells A2, B2, C2, D2....A12, B12, C12, D12 <o:p></o:p>

◻ Add 200 µl of fluorescein 1X stock solution into A1, B1, C1, D1 <o:p></o:p>

◻ Transfer 100 µl of fluorescein stock solution from A1 into A2. <o:p></o:p>

◻ Mix A2 by pipetting up and down 3x and transfer 100 µl into A3… <o:p></o:p>

◻ Mix A3 by pipetting up and down 3x and transfer 100 µl into A4... <o:p></o:p>

◻ Mix A4 by pipetting up and down 3x and transfer 100 µl into A5... <o:p></o:p>

◻ Mix A5 by pipetting up and down 3x and transfer 100 µl into A6... <o:p></o:p>

◻ Mix A6 by pipetting up and down 3x and transfer 100 µl into A7... <o:p></o:p>

◻ Mix A7 by pipetting up and down 3x and transfer 100 µl into A8... <o:p></o:p>

◻ Mix A8 by pipetting up and down 3x and transfer 100 µl into A9... <o:p></o:p>

◻ Mix A9 by pipetting up and down 3x and transfer 100 µl into A10... <o:p></o:p>

◻ Mix A10 by pipetting up and down 3x and transfer 100 µl into A11... <o:p></o:p>

◻ Mix A11 by pipetting up and down 3X and transfer 100 µl into liquid waste <o:p></o:p>

◻ Repeat dilution series for rows B, C, D <o:p></o:p>

◻ Measure fluorescence of all samples in all standard measurement modes in instrument <o:p></o:p>

◻ Record the data in your notebook <o:p></o:p>

◻ Import data into Excel (fluorescein standard curve tab ) Sheet_1 provided <o:p></o:p>

Cell measurement

Materials <o:p></o:p>

E.coli K-12 DH5α component cells <o:p></o:p>

LB (Luria Bertani) medium <o:p></o:p>

Chloramphenicol (stock concentration 30 mg/mL dissolved in EtOH - working stock 30 ug/mL) <o:p></o:p>

(This concentration refers to one of our PIs, and it’s the common concentration in my lab.) <o:p></o:p>

(The file can be download here: <o:p></o:p>

<a href="https://drive.google.com/open?id=0Bxb6U-RiSYsgbjEzU0o4eVNYNWc">https://drive.google.com/open?id=0Bxb6U-RiSYsgbjEzU0o4eVNYNWc</a> ) <o:p></o:p>

14 mL polypropylene round-bottom tube with aluminum foil cover <o:p></o:p>

(Comparing with 50mL falcon tube, it is much more suitable for incubation, the reason is that its cover isn’t completely closed which can make the oxygen entry freely, so the E.coli can be incubated in an aerobic environment.) <o:p></o:p>

Incubator at 37℃<o:p></o:p>

1.5 mL eppendorf tubes for sample storage<o:p></o:p>

Ice bucket with ice<o:p></o:p>

Pipettes<o:p></o:p>

Method<o:p></o:p>

Day 1: Transform E.coli DH5α with eight kinds of plasmids from 2017 iGEM kit, plate 7.<o:p></o:p>

Day 2: Pick 2 colonies from each of plate and inoculate it on 5 mL LB medium + chloramphenicol in 14mL polypropylene round-bottom tube. Grow the cells overnight for 18 hours at 37°C and 220 rpm. <o:p></o:p>

Day 3: Cell growth, sampling, and assay.<o:p></o:p>

◻ Set the instrument to read OD600 (as OD calibration setting)<o:p></o:p>

◻ Measure OD600 of the overnight cultures<o:p></o:p>

◻ Record data in the notebook<o:p></o:p>

◻ Import data into Excel (Dilution Calculation) Sheet_1 provided<o:p></o:p>

◻ Dilute the cultures to a target OD600 of 0.02 in 5 mL LB medium + Chloramphenicol 14mL polypropylene round-bottom tube with aluminum foil cover.<o:p></o:p>

(Overmuch cultures in tube leads to anaerobic environment. Therefore, about 1/3 cultures here.)<o:p></o:p>

◻ Incubate the cultures at 37°C and 220 rpm.<o:p></o:p>

◻ Take 500 µL samples of the cultures at 0, 2, 4, and 6 hours of incubation.<o:p></o:p>

◻ Place samples on ice.<o:p></o:p>

◻ At the end of sampling point, measure the samples (OD and Fl measurement).<o:p></o:p>

◻ Record data in the notebook.<o:p></o:p>

◻ Import data into Excel (cell measurement tab) Sheet_1 provided.<o:p></o:p>

Results

Calibration —— OD600 reference point

Raw data link: <a href="https://drive.google.com/open?id=0Bxb6U-RiSYsgTFlyeVRGeXlscGs">https://drive.google.com/open?id=0Bxb6U-RiSYsgTFlyeVRGeXlscGs</a><o:p></o:p>

Calibration —— fluorescein fluorescence standard curve

Raw data link: <a href="https://drive.google.com/open?id=0Bxb6U-RiSYsgVGl6RG1aQ1dxVFE">https://drive.google.com/open?id=0Bxb6U-RiSYsgVGl6RG1aQ1dxVFE</a><o:p></o:p>

According to the diagram above, we can notice that when the concentration of fluorescein is high (＞10μM), the curve doesn’t follow linear relation. As for this problem, we have communicated with other iGEM team, as expected, this is a common question. Referring to some materials, we give two possible explanations:<o:p></o:p>

——When concentration is too high, light cannot pass through the sample to cause excitation, thus very high concentrations can have very low fluorescence.<o:p></o:p>

——The surface portion of sample nearest the light absorbs too much light, little is available for the rest portion of the sample; thus the readings will not be linear, though the measurement will be within the range of a calibration curve.<o:p></o:p>

Cell measurement

Raw data link: <a href="https://drive.google.com/open?id=0Bxb6U-RiSYsgbnNJRVpOaUJLZVU">https://drive.google.com/open?id=0Bxb6U-RiSYsgbnNJRVpOaUJLZVU</a><o:p></o:p>

All the raw data and following tables refer to the wells arrangement of iGEM protocal below:<o:p></o:p>

Results:<o:p></o:p>

OD600 —— 0h<o:p></o:p>

OD600 —— 2h<o:p></o:p>

<img width="554" height="114" src="" v:shapes="图片_x0020_14"><o:p></o:p>

OD600 —— 4h<o:p></o:p>

<img width="554" height="116" src="" v:shapes="图片_x0020_15"><o:p></o:p>

OD600 —— 6h<o:p></o:p>

Fluorescence —— 0h<o:p></o:p>

<img width="554" height="115" src="" v:shapes="图片_x0020_17"><o:p></o:p>

Fluorescence —— 2h<o:p></o:p>

<img width="554" height="116" src="" v:shapes="图片_x0020_18"><o:p></o:p>

Fluorescence —— 4h<o:p></o:p>

<img width="554" height="115" src="" v:shapes="图片_x0020_19"><o:p></o:p>

Fluorescence —— 6h<o:p></o:p>

<img width="554" height="116" src="" v:shapes="图片_x0020_24"><o:p></o:p>

The curve are shown below:<o:p></o:p>

OD600 0——6 h<o:p></o:p>

We find that Positive Control and Test Device 1 have a lower OD600 value at 2h and 4h. According to the fluorescence curve, we suppose the more efficient GFP expression (strong promotor and RBS) leads to the less OD600 value. Generally speaking, all the eight kinds of bacteria have the similar growth condition during 0——6 h.<o:p></o:p>

Fluorescence 0——6 h<o:p></o:p>

<img width="420" height="630" src="" v:shapes="图片_x0020_21"><o:p></o:p>

According to the diagram above, we can find distinctly that Positive Control、 Test Device 1 and Test Device 4 have more efficient GFP expression than the others.<o:p></o:p>

Fluorescence/OD600 0——6 h<o:p></o:p>

Generally speaking, the fluorescence/OD600 value means the amount of GFP in each bacterium, thus, this value can represent the GFP expression strength than fluorescence value. <o:p></o:p>

the diagram shows the fluorescence/OD600 results among the six devices: <o:p></o:p>

Test Device 1 ＞ Test Device 2 ＞ Test Device 3 <o:p></o:p>

Test Device 4 ＞ Test Device 5 ＞ Test Device 6 <o:p></o:p>

The results correspond to the prediction, which means that our data is strict and reliable. <o:p></o:p>

   </div>

   <footer class="footer-distributed">

Companylogo

   </footer>

</body> <script> function openNav() {

   document.getElementById("mySidenav").style.width = "250px";

} openNav()

function closeNav() {

   document.getElementById("mySidenav").style.width = "0";

} </script> <style> .sidenav {

   height: 70%;
   width: 0;
   position: fixed;
   z-index: 1;
   top: 0;
   right: 0;
   background-color: #F4E6CF;

   overflow-x: hidden;
   transition: 0.5s;
   padding-top: 40px;
   margin: 200px 0px 200px 900px;

}

.sidenav a {

   /*侧边栏调整字体距离框的距离*/
   padding: 10px 50px 15px 100px;
   text-decoration: none;
   font-size: 20px;
   color: #818181;
   display: block;
   transition: 0.3s;

}

.sidenav a:hover {

   color: #f1f1f1;

}

.sidenav .closebtn {

   position: absolute;
   top: -15px;
   right: -30px;
   font-size: 15px;
   margin-left: 25px;

}

@media screen and (max-height: 450px) {

   .sidenav {
       padding-top: 15px;
   }
   .sidenav a {
       font-size: 10px;
   }

} </style>

</html>

Difference between revisions of "Team:Shanghaitech/InterLab"

Revision as of 12:00, 1 November 2017

Contents

Abstract

Plasmids and the prediction of results

All the eight plasmids are shown below:

Component cell strain & 96 well plate & Plate reader

Protocol

Cell measurement

Results

Calibration —— OD600 reference point

Calibration —— fluorescein fluorescence standard curve

Cell measurement

Results:<o:p></o:p>

Companylogo

@@ Line 2: / Line 2: @@
 {{Shanghaitech/css}}
 {{Shanghaitech/js}}
-<html lang="en">
-<head>
-    <meta http-equiv="content-type" content="text/html; charset=UTF-8">
-    <meta name="viewport" content="width=device-width, initial-scale=1, maximum-scale=1">
-    <meta http-equiv="X-UA-Compatible" content="ie=edge">
-    <title>Shanghaitech</title>
-    <meta name="description" content="">
-    <!-- HTML5 Shim and Respond.js 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/3.7.2/html5shiv.min.js"></script>
-    <script src="https://oss.maxcdn.com/respond/1.4.2/respond.min.js"></script>
-    <![endif]-->
-    <link rel="stylesheet" href="css/demo.css">
-    <link rel="stylesheet" href="css/footer-distributed-with-address-and-phones.css">
-    <link rel="stylesheet" href="http://maxcdn.bootstrapcdn.com/font-awesome/4.2.0/css/font-awesome.min.css">
-    <link href="http://fonts.googleapis.com/css?family=Cookie" rel="stylesheet" type="text/css">
-    <link rel="stylesheet" type="text/css" href="https://2017.igem.org/Template:Shanghaitech/CSS/DEMO?action=raw&ctype=text/css ">
-    <style type="text/css">
-    #menu-bar {
-        width: 100%;
-        margin: -1px 0px 0px -6px;
-        padding: 1.5px 0px 10px 260px;
-        height: 30px;
-        line-height: 100%;
-        border-radius: 0px;
-        -webkit-border-radius: 0px;
-        -moz-border-radius: 0px;
-        box-shadow: 0px 2px 3px #666666;
-        -webkit-box-shadow: 0px 2px 3px #666666;
-        -moz-box-shadow: 0px 2px 3px #666666;
-        background: url(https://static.igem.org/mediawiki/2017/thumb/0/07/T--Shanghaitech--nav.png/795px-T--Shanghaitech--nav.png);
-        border: solid 0px #6D6D6D;
-        position: fixed;
-        z-index: 999;
-    }
-    #menu-bar li {
-        margin: 0px 0px 10px 0px;
-        padding: 5px 5px 0px 6px;
-        float: left;
-        position: relative;
-        list-style: none;
-    }
-    #menu-bar a {
-        font-weight: bolder;
-        font-family: arial;
-        font-style: normal;
-        font-size: 15px;
-        color: #000000;
-        text-decoration: none;
-        display: block;
-        padding: 4px 19px 6px 16px;
-        margin: 0;
-        margin-bottom: 10px;
-        border-radius: 50px;
-        -webkit-border-radius: 50px;
-        -moz-border-radius: 50px;
-        text-shadow: 2px 2px 25px #3E17FF;
-    }
-    #menu-bar li ul li a {
-        margin: 0;
-    }
-    #menu-bar .active a,
-    #menu-bar li:hover>a {
-        /*菜单悬浮颜色*/
-        background: #D47272;
-        background: linear-gradient(top, #EB672F, #A1A1A1);
-        background: -ms-linear-gradient(top, #EB672F, #A1A1A1);
-        background: -webkit-gradient(linear, left top, left bottom, from(#EB672F), to(#A1A1A1));
-        background: -moz-linear-gradient(top, #EB672F, #A1A1A1);
-        color: #140044;
-        -webkit-box-shadow: 0 1px 1px rgba(0, 0, 0, .2);
-        -moz-box-shadow: 0 1px 1px rgba(0, 0, 0, .2);
-        box-shadow: 0 1px 1px rgba(0, 0, 0, .2);
-        text-shadow: 0px 0px 0px #FFFFFF;
-    }
-    #menu-bar ul li:hover a,
-    #menu-bar li:hover li a {
-        background: none;
-        border: none;
-        color: #666;
-        -box-shadow: none;
-        -webkit-box-shadow: none;
-        -moz-box-shadow: none;
-    }
-    #menu-bar ul a:hover {
-        /*子菜单悬浮颜色*/
-        background: #0399D4 !important;
-        background: linear-gradient(top, #346BEC, #0186BA) !important;
-        background: -ms-linear-gradient(top, #346BEC, #0186BA) !important;
-        background: -webkit-gradient(linear, left top, left bottom, from(#346BEC), to(#0186BA)) !important;
-        background: -moz-linear-gradient(top, #346BEC, #0186BA) !important;
-        color: #000000 !important;
-        border-radius: 0;
-        -webkit-border-radius: 0;
-        -moz-border-radius: 0;
-        text-shadow: 2px 2px 9px #FFFFFF;
-    }
-    #menu-bar li:hover>ul {
-        display: block;
-    }
-    #menu-bar ul {
-        background: #DDDDDD;
-        background: linear-gradient(top, #FFFBC4, #CFCFCF);
-        background: -ms-linear-gradient(top, #FFFBC4, #CFCFCF);
-        background: -webkit-gradient(linear, left top, left bottom, from(#FFFBC4), to(#CFCFCF));
-        background: -moz-linear-gradient(top, #FFFBC4, #CFCFCF);
-        display: none;
-        margin: 0;
-        padding: 0;
-        width: 185px;
-        position: absolute;
-        top: 33px;
-        left: 0;
-        border: solid 1px #B4B4B4;
-        border-radius: 10px;
-        -webkit-border-radius: 10px;
-        -moz-border-radius: 10px;
-        -webkit-box-shadow: 0px 2px 3px #222222;
-        -moz-box-shadow: 0px 2px 3px #222222;
-        box-shadow: 0px 2px 3px #222222;
-    }
-    #menu-bar ul li {
-        float: none;
-        margin: 0;
-        padding: 0;
-    }
-    #menu-bar ul a {
-        padding: 10px 0px 10px 15px;
-        color: #424242 !important;
-        font-size: 11px;
-        font-style: normal;
-        font-family: arial;
-        font-weight: bold;
-        text-shadow: 2px 2px 3px #FFFFFF;
-    }
-    #menu-bar ul li:first-child>a {
-        border-top-left-radius: 10px;
-        -webkit-border-top-left-radius: 10px;
-        -moz-border-radius-topleft: 10px;
-        border-top-right-radius: 10px;
-        -webkit-border-top-right-radius: 10px;
-        -moz-border-radius-topright: 10px;
-    }
-    #menu-bar ul li:last-child>a {
-        border-bottom-left-radius: 10px;
-        -webkit-border-bottom-left-radius: 10px;
-        -moz-border-radius-bottomleft: 10px;
-        border-bottom-right-radius: 10px;
-        -webkit-border-bottom-right-radius: 10px;
-        -moz-border-radius-bottomright: 10px;
-    }
-    #menu-bar:after {
-        content: ".";
-        display: block;
-        clear: both;
-        visibility: hidden;
-        line-height: 0;
-        height: 0;
-    }
-    #menu-bar {
-        display: inline-block;
-    }
-    html[xmlns] #menu-bar {
-        display: block;
-    }
-    * html #menu-bar {
-        height: 1%;
-    }
-    </style>
-    <style type="text/css">
-        .content {
-        background-color: rgba(255, 255, 255, 0.90);
-        width: 73%;
-        border: 1px solid #cccccc;
-        border-radius: 10px;
-        box-shadow: 1px 1px 3px rgba(0, 0, 0, 0.35);
-        color: #3d3d3d;
-        font-size: 18px;
-        margin-left: 2%;
-        margin-top: 50px;
-        text-align: justify;
-        padding: 10px 70px 30px 70px;
-    </style>
-    <style type="text/css">
-    </style>
-</head>
 <body>