Difference between revisions of "Team:NAU-CHINA/InterLab"

(Undo revision 288050 by WEISIQI (talk))
Line 1: Line 1:
−
    {{NAU-CHINA/ResetCSS}}
+
{{NAU-CHINA/ResetCSS}}
 
{{NAU-CHINA}}
 
{{NAU-CHINA}}
 
{{NAU-CHINA/header}}
 
{{NAU-CHINA/header}}
Line 79: Line 79:
 
         <p>InterLab</p>
 
         <p>InterLab</p>
 
     </div>-->   
 
     </div>-->   
−
 
+
   
 
     <div class="main-content">
 
     <div class="main-content">
 
         <div class="textblock">
 
         <div class="textblock">
−
             <h1>Overview </h1>          
+
             <h1>Introduction</h1>
−
             <p>Regarding the planning of this HP-related event, we have conceived a more unique idea - the development and production of iGEM's exclusive app which we call iGEMCloud.At present,all the information about iGEM in China's participating regions comes from: QQ group, WeChat group, Post bar, Zhihu, iGEM official website, etc. The information about the events that these platforms can get is more scattered and in a certain The timeliness, authenticity, and referability of the information obtained on these platforms are not guaranteed.</p>
+
             <p>Reliable and repeatable measurement is a key component to all engineering disciplines. Based on this idea, the measurement committee encourages all competing teams to participate in the InterLab study. This year, the committee wants iGEMers from around the world to answer the following question together: Can we reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of OD?</p>
 
         </div>
 
         </div>
−
    </div>
+
 
−
    <div class="main-content">
+
 
         <div class="textblock">
 
         <div class="textblock">
−
             <h1>Instructions of usage</h1>          
+
             <h1>Materials</h1>
−
             <p>1.Register an account with your email address,enter the verify code,and use this address to log in.</p>
+
             <p>
−
            <p>2.In the application, you can receive official news, notifications, etc. so that you can get the latest news of iGEM events in a timely, convenient and accurate manner.</p>
+
                1.Measurement Kit (provided with the iGEM distribution shipment) containing:<br>
−
            <p>3.Users can write blog posts in the discussion forum for all users to browse. The blog post is in the form of pictures + text. The blog posts are classified into different sections, such as experiment, seek help, etc.so that you can share your ideas with all iGEM participants.<br>
+
                1ml LUDOX CL-X<br>
−
            Excellent blog posts will be tagged so that they are discovered by users.At the same time, users who see the blog post written by you can comment or reply to you. The content of the reply is in the form of image + text.when you find a nice blog post, click on the star symbol in the upper right to bookmark it for later reading.</p>
+
                150 μL Silica Bead (microsphere suspension)<br>
−
            <p>4.If you want to remind specific users to view the content you posted, you can remind the user to view this blog post or comment by "@" + "user nickname".</p>
+
                Fluorescein (powder, in amber tube)<br>
−
            <p>5.You can search for blog posts in one or more keywords in the app, and sort the search results by time, number of view , and collection. You can also search for users or teams.</p>
+
                2. iGEM Parts Distribution Kit Plates<br>
−
            <p>6.You can follow the teams or users that you think are worthy of attention so that you can view their published content more easily in the future.</p>
+
                3. 1x PBS (phosphate buffered saline, pH 7.4 – 7.6)<br>
−
            <p>7.Each user can create or join a team, become a member, and use their team space, the team creator is the captain. The captain can edit the team information in the team space to introduce others to the team and show the team research direction.Only members of the team can post in the team space, but everyone can view it, the captain can also add albums in the team space, members of the team can post photos in the album.</p>
+
                4. ddh1O (ultrapure filtered or double distilled water)<br>
−
             <p>8.This app has built-in gadgets that may be needed for the lab and more other features will be added in the future.......</p>
+
                5. Competent cells (Escherichia coli strain DH5α)<br>
 +
                6. LB (Luria Bertani) media<br>
 +
                7. Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH)<br>
 +
                8. 50 ml Falcon tube (covered in foil to block light)<br>
 +
                9. Incubator at 37°C<br>
 +
                10.  Plate reader (SpectraMax i3x)<br>
 +
                11. 1.5 ml eppendorf tubes<br>
 +
                12.  Ice bucket with ice<br>
 +
                Micropipettes (capable of pipetting a range of volumes between 1 μL and 1000 μL)<br>
 +
                Micropipette tips<br>
 +
                96 well plates, black with clear flat bottom<br>
 +
             </p>
 
         </div>
 
         </div>
  
−
    </div>
 
−
 
−
    <div class="main-content">
 
 
         <div class="textblock">
 
         <div class="textblock">
−
             <h1>How to get it</h1>    
+
             <h1>Calibration</h1>
 +
            <div class="section">
 +
                <h2>1. OD600 Reference point</h2>
 +
                <p>
 +
                    We used LUDOX CL-X (45% colloidal silica suspension) as a single point reference to obtain a conversion factor to transform the absorbance (Abs600) data from our plate reader into a comparable OD600 measurement as would be obtained in a spectrophotometer.
 +
                </p>
 +
                <figure>
 +
                    <figcaption class="_table">Table 1. The data for OD600 measured by our plate reader, with a ratiometric conversion factor of 3.505.</figcaption>
 +
                    <img src="https://static.igem.org/mediawiki/2018/9/9e/T--NAU-China--interlab6.png">
 +
                </figure>
 +
            </div>
 +
 
 +
            <div class="section">
 +
                <h2>2. Particle Standard Curve</h2>
  
−
            <p>Scan the QR code to get the application.</p>
+
                <p>We prepared a dilution series of monodisperse silica microspheres as instructed in the protocol.</p>
−
            <figure>      
+
                <figure>
−
                     <img src="https://static.igem.org/mediawiki/2018/2/2c/T--NAU-China--software1.png">
+
                     <img src="https://static.igem.org/mediawiki/2018/f/fd/T--NAU-China--interlab1.jpg">
 +
                    <figcaption>Figure 1. Serial dilution of monodisperse silica microspheres.</figcaption>
 
                 </figure>
 
                 </figure>
−
         
+
 
 +
                <p>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.</p>
 +
                <figure>
 +
                    <figcaption class="_table">Table 2. The data for Abs600 measured by our plate reader.</figcaption>
 +
                    <img src="https://static.igem.org/mediawiki/2018/1/15/T--NAU-China--interlab7.png">
 +
                </figure>
 +
 
 +
                <figure>
 +
                    <img src="https://static.igem.org/mediawiki/2018/4/4c/T--NAU-China--interlab8.jpg">
 +
                    <figcaption>Figure 2-1. Abs600 is plotted against the particle count.</figcaption>
 +
                </figure>
 +
                <figure>
 +
                    <img src="https://static.igem.org/mediawiki/2018/9/9a/T--NAU-China--interlab9.jpg">
 +
                    <figcaption>Figure 2-2. Abs600 is plotted against the particle count on a logarithmic scale.</figcaption>
 +
                </figure>
 +
 
 +
                <figure>
 +
                    <figcaption class="_table">Table 3. The data for particles/Abs600.</figcaption>
 +
                    <img src="https://static.igem.org/mediawiki/2018/4/4d/T--NAU-China--interlab10.jpg">
 +
                </figure>
 +
            </div>
 +
 
 +
 
 +
            <div>
 +
                <h2>3. Fluorescence standard curve</h2>
 +
 
 +
                <p>We prepared a dilution series of fluorescein as instructed in the protocol. </p>
 +
                <figure>
 +
                    <img src="https://static.igem.org/mediawiki/2018/thumb/f/f5/T--NAU-China--interlab2.jpg/1200px-T--NAU-China--interlab2.jpg">
 +
                    <figcaption>Figure 3. Serial dilution of monodisperse silica fluorescein.</figcaption>
 +
                </figure>
 +
                <p>
 +
                    By measuring the serial dilutions in our plate reader, we generated a standard curve of fluorescence for fluorescein concentration. We were able to use this to convert our cell based readings to an equivalent fluorescein concentration.
 +
                </p>
 +
                <figure>
 +
                    <figcaption class="_table">Table 4. The data for fluorescence measured by our plate reader.</figcaption>
 +
 
 +
                   
 +
                    <img src="https://static.igem.org/mediawiki/2018/7/77/T--NAU-China--interlab11.jpg">
 +
                </figure>
 +
 
 +
                <figure>
 +
 
 +
                    <img src="https://static.igem.org/mediawiki/2018/b/ba/T--NAU-China--interlab12.jpg">
 +
                   
 +
 
 +
                    <figcaption>Figure 4-1. Fluorescence is plotted against the fluorescein concentration.</figcaption>
 +
                </figure>
 +
                <figure>
 +
 
 +
                    <img src="https://static.igem.org/mediawiki/2018/5/56/T--NAU-China--interlab13.jpg">
 +
 
 +
                   
 +
 
 +
                    <figcaption>Figure 4-2. Fluorescence is plotted against the fluorescein concentration on a logarithmic scale.</figcaption>
 +
                </figure>
 +
                <figure>
 +
                    <figcaption class="_table">Table 5. The data for fluorescein/a.u..</figcaption>               
 +
                    <img src="https://static.igem.org/mediawiki/2018/7/70/T--NAU-China--interlab14.jpg">
 +
                </figure>
 +
            </div>
 +
 
 +
            <h1>Cell Measurement</h1>
 +
 
 +
            <div>
 +
                <h2>Transformation</h2>
 +
               
 +
                <p>We transformed the plasmids (listed below) resuspended from the Distribution Kit into E. coli DH5α cells. DH5α competent cells were bought from Transgen Biotech. The transformation was successful for all the plasmids and resulted in a sufficient amount of colonies on all plates.</p>
 +
                <figure>
 +
                    <figcaption class="_table">Table 6. The plasmids used for transformation.</figcaption>                   
 +
                    <img src="https://static.igem.org/mediawiki/2018/e/eb/T--NAU-China--interlab15.jpg">
 +
                </figure>
 +
            </div>
 
         </div>
 
         </div>
−
    </div>
 
  
−
    <div class="main-content">
 
 
         <div class="textblock">
 
         <div class="textblock">
−
            <h1>What it could be:</h1>
 
 
             <div class="section">
 
             <div class="section">
−
                 <h2>A information gathering platform:</h2>
+
                 <h2>Cell Culture</h2>              
−
                 <p>We will gather information from other website to release,and we hope to build contact with some organization to get instant and accurate information.The user of this app can share their information in it,if it is valuable and reliable,we will push it to everyone.</p>
+
                 <p>We picked 2 colonies from each of the transformation plates and inoculate in 5 mL LB medium + Chloramphenicol. The cells were grown overnight 16 hours at 37°C and 220 rpm.</p>
 
             </div>
 
             </div>
−
            <div class="section">
+
        </div>
−
                 <h2>A platform for interaction between teams:</h2>
+
 
−
                 <p>Through the form of the team space,each team can easily interact with other teams.This will strengthen the connection between iGEM teams and produce countless positive possibilities.</p>
+
        <div class="textblock">
 +
            <div class="section">
 +
                 <h2>Sampling</h2>
 +
               
 +
                 <p>We made a 1:10 dilution of each overnight culture in LB + Chloramphenicol and measured Abs600 of these 1:10 diluted cultures. After measuring, we further diluted these cultures to a target Abs600 of 0.02 in a final volume of 12 ml LB medium + Chloramphenicol in 50 mL falcon tubes. These falcon tubes were covered in foil to block light. We took 500 µL samples of the diluted cultures at 0 hours into 1.5 ml eppendorf tubes prior to incubation. These samples were put on ice before measurement was taken. Then we incubated the remainder of the cultures at 37°C and 220 rpm for 6 hours. After these procedures were finished, we took 500 µL samples of the cultures at 6 hours of incubation into 1.5 ml eppendorf tubes and placed them on ice. At the end, we carried out our measurement. Samples were laid out in 96 well plates according to the diagram below:</p>
 +
                <figure>
 +
                    <img src="https://static.igem.org/mediawiki/2018/a/a8/T--NAU-China--interlab3.jpg">
 +
                    <figcaption>Figure 5. Samples were laid out in 96 well plates as shown.</figcaption>
 +
                </figure>
 +
               
 +
                <figure>
 +
                    <figcaption class="_table">Table 7-1. Fluorescence raw readings at hour 0.</figcaption>                   
 +
                    <img src="https://static.igem.org/mediawiki/2018/0/03/T--NAU-China--interlab16.jpg">
 +
                </figure>
 +
               
 +
                <figure>
 +
                    <figcaption class="_table">Table 7-2. Abs600 raw readings at hour 0.</figcaption>                   
 +
                    <img src="https://static.igem.org/mediawiki/2018/2/28/T--NAU-China--interlab17.jpg">
 +
                </figure>
 +
               
 +
                <figure>
 +
                    <figcaption class="_table">Table 7-3. Fluorescence raw readings at hour 6.</figcaption>                   
 +
                    <img src="https://static.igem.org/mediawiki/2018/4/47/T--NAU-China--interlab18.jpg">
 +
                </figure>
 +
               
 +
                <figure>
 +
                    <figcaption class="_table">Table 7-4. Abs600 raw readings at hour 6.</figcaption>                   
 +
                    <img src="https://static.igem.org/mediawiki/2018/f/fe/T--NAU-China--interlab19.jpg">
 +
                </figure>
 +
               
 +
                <p>After some calculation, we got some experimental values on uM Fluorescein / OD:</p>
 +
                <figure>
 +
                    <figcaption class="_table">Table 8-1. uM Fluorescein / OD at hour 0.</figcaption>                   
 +
                    <img src="https://static.igem.org/mediawiki/2018/7/70/T--NAU-China--interlab20.jpg">
 +
                </figure>
 +
               
 +
                <figure>
 +
                    <figcaption class="_table">Table 8-2. uM Fluorescein / OD at hour 6.</figcaption>                   
 +
                    <img src="https://static.igem.org/mediawiki/2018/d/d1/T--NAU-China--interlab21.jpg">
 +
                </figure>
 
             </div>
 
             </div>
−
            <div class="section">
+
            <div class="section">
−
                 <h2>An iGEM community:</h2>
+
                 <h2>CFU</h2>
−
                 <p>Each iGEMer could leave a footprint here,show themselves here.Everyone here is part of the iGEM.</p>
+
               
−
                 <p>The App we conceived can provide comprehensive information on the release of events, each team to register their own team home page, various activities related to iGEM, question answer and experience sharing , topic discussion, experimental supplies, and more.
+
                 <p>In this part, we want to know whether OD600 can be calibrated to colony forming unit (CFU) counts. We counted two Positive Control (BBa_I20270) cultures and two Negative Control (BBa_R0040) cultures.</p>
−
</p>
+
                 <p>We first diluted our overnight culture to the linear detection range of our plate reader by adding 25 μL culture to 175 μL LB + Cam in a well in a black 96-well plate. Then we further diluted them to OD600 = 0.1 in 1mL of LB + Cam media and did this in triplicate for each culture.</p>
 +
 
 +
                <figure>
 +
                    <img src="https://static.igem.org/mediawiki/2018/b/b7/T--NAU-China--interlab4.jpg">               
 +
                    <figcaption>Figure 6. Check whether the OD600 is 0.1 (minus the blank measurement).</figcaption>
 +
                </figure>
 +
 
 +
                <p>After ensuring all the OD600 is 0.1, we performed serial dilutions and incubation for them as directed.</p>
 +
                <figure>
 +
                    <img src="https://static.igem.org/mediawiki/2018/b/b7/T--NAU-China--interlab5.jpg">
 +
                    <figcaption>Figure 7. Serial dilutions and plates for these samples.</figcaption>
 +
                </figure>
 +
 
 +
                <p>Here are our data:</p>
 +
                <img src="https://static.igem.org/mediawiki/2018/1/1d/T--NAU-China--interlab22.jpg">
 
             </div>
 
             </div>
 +
 +
        <div class="textblock">
 +
            <h1>Discussion</h1>           
 +
            <p>
 +
                We did our experiments twice. The first attempt was not so successful as the bacteria grew faster than we assumed. We were not able to keep them at a target OD. Our laboratory is on the first floor, while the plate reader is on the sixth floor. During this period, when we read the number from the machine and ran upstairs to do dilution, the OD had already changed a lot. So we recommend using an ice pack to slow down cell growth in order to obtain better data. All the dilutions and transporting may be performed on ice, just as we did in our second attempt. Additionally, we were not so optimistic about the results of serial dilution and CFU counting as the dilution may not be so accurate, and are easy to be affected by complicated reasons. Our data still weren’t look so perfect although we made every step quite carefully and did a second try.
 +
            </p>
 
         </div>
 
         </div>
 
     </div>
 
     </div>
−
   
 
−
     
 
 
</body>
 
</body>
 
</html>
 
</html>

Revision as of 06:59, 14 October 2018

Template:2018_NAU-CHINA

header
InterLab

Introduction

Reliable and repeatable measurement is a key component to all engineering disciplines. Based on this idea, the measurement committee encourages all competing teams to participate in the InterLab study. This year, the committee wants iGEMers from around the world to answer the following question together: Can we reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of OD?

Materials

1.Measurement Kit (provided with the iGEM distribution shipment) containing:
1ml LUDOX CL-X
150 μL Silica Bead (microsphere suspension)
Fluorescein (powder, in amber tube)
2. iGEM Parts Distribution Kit Plates
3. 1x PBS (phosphate buffered saline, pH 7.4 – 7.6)
4. ddh1O (ultrapure filtered or double distilled water)
5. Competent cells (Escherichia coli strain DH5α)
6. LB (Luria Bertani) media
7. Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH)
8. 50 ml Falcon tube (covered in foil to block light)
9. Incubator at 37°C
10. Plate reader (SpectraMax i3x)
11. 1.5 ml eppendorf tubes
12. Ice bucket with ice
Micropipettes (capable of pipetting a range of volumes between 1 μL and 1000 μL)
Micropipette tips
96 well plates, black with clear flat bottom

Calibration

1. OD600 Reference point

We used LUDOX CL-X (45% colloidal silica suspension) as a single point reference to obtain a conversion factor to transform the absorbance (Abs600) data from our plate reader into a comparable OD600 measurement as would be obtained in a spectrophotometer.

Table 1. The data for OD600 measured by our plate reader, with a ratiometric conversion factor of 3.505.

2. Particle Standard Curve

We prepared a dilution series of monodisperse silica microspheres as instructed in the protocol.

Figure 1. Serial dilution of monodisperse silica microspheres.

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.

Table 2. The data for Abs600 measured by our plate reader.
Figure 2-1. Abs600 is plotted against the particle count.
Figure 2-2. Abs600 is plotted against the particle count on a logarithmic scale.
Table 3. The data for particles/Abs600.

3. Fluorescence standard curve

We prepared a dilution series of fluorescein as instructed in the protocol.

Figure 3. Serial dilution of monodisperse silica fluorescein.

By measuring the serial dilutions in our plate reader, we generated a standard curve of fluorescence for fluorescein concentration. We were able to use this to convert our cell based readings to an equivalent fluorescein concentration.

Table 4. The data for fluorescence measured by our plate reader.
Figure 4-1. Fluorescence is plotted against the fluorescein concentration.
Figure 4-2. Fluorescence is plotted against the fluorescein concentration on a logarithmic scale.
Table 5. The data for fluorescein/a.u..

Cell Measurement

Transformation

We transformed the plasmids (listed below) resuspended from the Distribution Kit into E. coli DH5α cells. DH5α competent cells were bought from Transgen Biotech. The transformation was successful for all the plasmids and resulted in a sufficient amount of colonies on all plates.

Table 6. The plasmids used for transformation.

Cell Culture

We picked 2 colonies from each of the transformation plates and inoculate in 5 mL LB medium + Chloramphenicol. The cells were grown overnight 16 hours at 37°C and 220 rpm.

Sampling

We made a 1:10 dilution of each overnight culture in LB + Chloramphenicol and measured Abs600 of these 1:10 diluted cultures. After measuring, we further diluted these cultures to a target Abs600 of 0.02 in a final volume of 12 ml LB medium + Chloramphenicol in 50 mL falcon tubes. These falcon tubes were covered in foil to block light. We took 500 µL samples of the diluted cultures at 0 hours into 1.5 ml eppendorf tubes prior to incubation. These samples were put on ice before measurement was taken. Then we incubated the remainder of the cultures at 37°C and 220 rpm for 6 hours. After these procedures were finished, we took 500 µL samples of the cultures at 6 hours of incubation into 1.5 ml eppendorf tubes and placed them on ice. At the end, we carried out our measurement. Samples were laid out in 96 well plates according to the diagram below:

Figure 5. Samples were laid out in 96 well plates as shown.
Table 7-1. Fluorescence raw readings at hour 0.
Table 7-2. Abs600 raw readings at hour 0.
Table 7-3. Fluorescence raw readings at hour 6.
Table 7-4. Abs600 raw readings at hour 6.

After some calculation, we got some experimental values on uM Fluorescein / OD:

Table 8-1. uM Fluorescein / OD at hour 0.
Table 8-2. uM Fluorescein / OD at hour 6.

CFU

In this part, we want to know whether OD600 can be calibrated to colony forming unit (CFU) counts. We counted two Positive Control (BBa_I20270) cultures and two Negative Control (BBa_R0040) cultures.

We first diluted our overnight culture to the linear detection range of our plate reader by adding 25 μL culture to 175 μL LB + Cam in a well in a black 96-well plate. Then we further diluted them to OD600 = 0.1 in 1mL of LB + Cam media and did this in triplicate for each culture.

Figure 6. Check whether the OD600 is 0.1 (minus the blank measurement).

After ensuring all the OD600 is 0.1, we performed serial dilutions and incubation for them as directed.

Figure 7. Serial dilutions and plates for these samples.

Here are our data:

Discussion

We did our experiments twice. The first attempt was not so successful as the bacteria grew faster than we assumed. We were not able to keep them at a target OD. Our laboratory is on the first floor, while the plate reader is on the sixth floor. During this period, when we read the number from the machine and ran upstairs to do dilution, the OD had already changed a lot. So we recommend using an ice pack to slow down cell growth in order to obtain better data. All the dilutions and transporting may be performed on ice, just as we did in our second attempt. Additionally, we were not so optimistic about the results of serial dilution and CFU counting as the dilution may not be so accurate, and are easy to be affected by complicated reasons. Our data still weren’t look so perfect although we made every step quite carefully and did a second try.