Difference between revisions of "Team:FJNU-China/Description"

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  <h1 style="text-align: center;margin-top: 100px; font-size:35px;">Description</h1>
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  <h1 style="text-align: center;margin-top: 100px;">Description</h1>
 
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            <h2 >Overview</h2>
 +
            <p>&nbsp;&nbsp;Reliable and repeatable measurement is a key component to all engineering disciplines. However, the number of cells in the sample is a  variability in measurements. The goal of the iGEM InterLab Study is to identify and correct the sources of systematic variability in synthetic biology measurements. This year, our team take part in the fifth InterLab study, using <span style="font-style:italic;">normalizing</span> to absolute cell count or CFUs instead of OD to reduce variability of fluorescence measurements.</p>
 +
            <hr>
 +
            <h2> Materials</h2>
 +
            <p ><span style="font-size:20px;font-weight:bold;">Strain used</span></br><span style=" font-style:italic;">Escherichia coli</span>&nbsp;&nbsp;DH5α</p>
 +
            <p ><span style="font-size:20px;font-weight:bold;">Plasmid used</span></br>Negative control: BBa_R0040  Plate 7 Well 2D </br>
 +
Positive control: BBa_I20270  Plate 7 Well 2B  </br>
 +
Test Device 1: BBa_J364000  Plate 7 Well 2F  </br>
 +
Test Device 2: BBa_J364001  Plate 7 Well 2H  </br>
 +
Test Device 3: BBa_J364002  Plate 7 Well 2J  </br>
 +
Test Device 4: BBa_J364007  Plate 7 Well 2L  </br>
 +
Test Device 5: BBa_J364008  Plate 7 Well 2N  <br>
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Test Device 6: BBa_J364009  Plate 7 Well 2P
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</p>
 +
            <p ><span style="font-size:20px;font-weight:bold;">Machine</span></br>Molecular Devices SpectraMax i3x
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</p>
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            <hr>
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 +
            <h2 >Methods</h2>
 +
            <p>&nbsp;&nbsp;We followed this <a href="https://static.igem.org/mediawiki/2018/3/3e/T--FJNU-China--Interlab-Protocol.pdf">protocol</a> to do the Interlab Study.</br>&nbsp;&nbsp;When we completed three of the calibration measurements, performing the cell measurements. Used the same plates, volumes and settings that we used in calibration protocol.  We transformed E.coli DH5α competent cells with the 8 plasmids and picked 2 colonies from each of plates into 5 mL LB medium + Chloramphenicol. After culturing the cells overnight at 37°C and 220 rpm, we used plate reader to measure the Abs600 and fluorescence of samples at 0, 6 hours.</p>
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            <hr>
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 +
            <h2 >Result</h2>
 +
          <p ><span style="font-size:20px;font-weight:bold;">Calibration</span></br>Calibration 1: OD600 reference point</p>
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    <div>
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          </br><p >Calibration 2: Particle Standard Curve</p>
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    <li>Abstract</li>
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<div class="tz-gallery">
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    <li>PLA</li>
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    <li>2-PE</li>
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    <li>Bromidrosis</li>
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    <li>Biosafty</li>
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<h2 class="target" style="color: tomato;">Abstract</h2>
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<img src="https://static.igem.org/mediawiki/2018/d/de/T--FJNU-China--Interlab-figue12.png" alt="table1">
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  <p>As we know, the purpose of modeling is mainly to provide guidance and solve problems for the team experiment. So, before we designed the model, we sorted out some of the problems the team was facing. Based on these three problems,.Because The effect of PLA concentration on the quantity Staphylococcus epidermidis.Vestibulum quis quam ut magna consequat faucibus. Pellentesque eget nisi a mi suscipit tincidunt. Ut tempus dictum risus. Pellentesque viverra sagittis quam at mattis. Suspendisse potenti. Aliquam sit amet gravida nibh, facilisis gravida odio. Phasellus auctor velit at lacus blandit, commodo iaculis justo viverra. Etiam vitae est arcu.</p>
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<h2 class="target" style="color: tomato;">PLA</h2>
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  <p>As we know, the purpose of modeling is mainly to provide guidance and solve problems for the team experiment. So, before we designed the model, we sorted out some of the problems the team was facing. Based on these three problems,.Because The effect of PLA concentration on the quantity Staphylococcus epidermidis.Vestibulum quis quam ut magna consequat faucibus. Pellentesque eget nisi a mi suscipit tincidunt. Ut tempus dictum risus. Pellentesque viverra sagittis quam at mattis. Suspendisse potenti. Aliquam sit amet gravida nibh, facilisis gravida odio. Phasellus auctor velit at lacus blandit, commodo iaculis justo viverra. Etiam vitae est arcu.</p>
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<a class="lightbox" href="https://static.igem.org/mediawiki/2018/e/ee/T--FJNU-China--Interlab-figue34.png">
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          <p ><span style="font-size:20px;font-weight:bold;">Cell measurement</span></p>
  
  
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  <p>As we know, the purpose of modeling is mainly to provide guidance and solve problems for the team experiment. So, before we designed the model, we sorted out some of the problems the team was facing. Based on these three problems,.Because The effect of PLA concentration on the quantity Staphylococcus epidermidis.Vestibulum quis quam ut magna consequat faucibus. Pellentesque eget nisi a mi suscipit tincidunt. Ut tempus dictum risus. Pellentesque eget nisi a mi suscipit tincidunt. Ut tempus dictum risus. </p>
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<img src="https://static.igem.org/mediawiki/2018/b/bc/T--FJNU-China--pick.png" style="width: 100%"><img src="https://static.igem.org/mediawiki/2018/b/bc/T--FJNU-China--pick.png" style="width: 100%">
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<div class="col-sm-12 col-md-12">
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<h2 class="target" style="color: tomato;">Garbage can</h2>
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<a class="lightbox" href="https://static.igem.org/mediawiki/2018/3/35/T--FJNU-China--Interlab-table2.png">
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<div >
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<img src="https://static.igem.org/mediawiki/2018/3/35/T--FJNU-China--Interlab-table2.png">
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  <p>As we know, the purpose of modeling is mainly to provide guidance and solve problems for the team experiment. So, before we designed the model, we sorted out some of the problems the team was facing. Based on these three problems,.Because The effect of PLA concentration on the quantity Staphylococcus epidermidis.Vestibulum quis quam ut magna consequat faucibus. Pellentesque eget nisi a mi suscipit tincidunt. Ut tempus dictum risus. Pellentesque viverra sagittis quam at mattis. Suspendisse potenti. Aliquam sit amet gravida nibh, facilisis gravida odio. Phasellus auctor velit at lacus blandit, commodo iaculis justo viverra. Etiam vitae est arcu.</p>
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</a>
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<a class="lightbox" href="https://static.igem.org/mediawiki/2018/5/56/T--FJNU-China--Interlab-figue56.png">
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<img src="https://static.igem.org/mediawiki/2018/5/56/T--FJNU-China--Interlab-figue56.png" alt="figue56">
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</a>
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</div>
  
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<h2 class="target" style="color: tomato;">Bromidrosis</h2>
 
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<div>
 
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  <p>As we know, the purpose of modeling is mainly to provide guidance and solve problems for the team experiment. So, before we designed the model, we sorted out some of the problems the team was facing. Based on these three problems,.Because The effect of PLA concentration on the quantity Staphylococcus epidermidis.Vestibulum quis quam ut magna consequat faucibus. Pellentesque eget nisi a mi suscipit tincidunt. Ut tempus dictum risus. Pellentesque viverra sagittis quam at mattis. Suspendisse potenti. Aliquam sit amet gravida nibh, facilisis gravida odio. Phasellus auctor velit at lacus blandit, commodo iaculis justo viverra. Etiam vitae est arcu.</p>
 
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  <img src="https://static.igem.org/mediawiki/2018/b/ba/T--FJNU-China--on_skin.gif" style="width: 80%;"> <img src="https://static.igem.org/mediawiki/2018/7/79/T--FJNU-China--room-temperature.gif" style="width: 80%;">  <img src="https://static.igem.org/mediawiki/2018/b/bc/T--FJNU-China--pick.png" style="width: 45%;">  <img src="https://static.igem.org/mediawiki/2018/b/bc/T--FJNU-China--pick.png" style="width: 45%;"> <img src="https://static.igem.org/mediawiki/2018/b/bc/T--FJNU-China--pick.png" style="width: 45%;">
 
 
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<h2 class="target" style="color: tomato;">Biosafty</h2>
 
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<div>
 
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<h3 style="text-align:left;"><i class="fa fa-hand-o-right"  style=" align:center; color:tomato;" ></i>&nbsp;&nbsp;Background</h3>
 
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  <p>mazEF is a toxin-antitoxin module located on the Escherichia coli chromosome and that of some other bacteria, including pathogens. mazF specifies for a stable toxin, MazF, and mazE specifies for a labile antitoxin, MazE, that antagonizes MazF. (Engelberg-Kulka, H., et al. 2005)MazF is a toxic nuclease arresting cell growth through the mechanism of RNA cleavage and that MazE inhibits the RNase activity of MazF by forming a complex.(Syed, M. A., et al. 2011).We used mazF as our killer to ensure biosafty. </p>
 
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</div>
 
</div>
  
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<h3 style="text-align:left;"><i class="fa fa-hand-o-right"  style=" align:center; color:tomato;" ></i>&nbsp;&nbsp;Design</h3>
 
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  <p>According to different scenarios, we designed different promoters to combine with mazF. With the humanization design, the biosafty of our projects is guaranteed. </p>
 
  
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<h3 style="text-align:left;">&nbsp;&nbsp;&nbsp;&nbsp;Bromidrosis</h3>
 
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  <p>In our scent spray, we designed a suicide switch induced by arabinose. When we don't want the engineered bacteria to work again, we can kill the engineered bacteria by spraying a spray containing arabinose. In order to ensure that our engineering bacteria do not work in other environments, we set the switch of PLA and 2-PE to salt control and temperature control. Only when the temperature reaches the body temperature condition and the salinity reaches the human sweating condition, our engineering bacteria will play a role. </p>
 
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<h3 style="text-align:left;">&nbsp;&nbsp;&nbsp;&nbsp;Garbage can</h3>
 
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  <p>In the trash can lid, we chose a light-controlled promoter to combine with mazF. Once the engineered bacteria leaves the dark environment of the trash can, it will be killed. </p>
 
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            <hr>
  
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            <h2 >Analyse</h2>
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            <p style="margin-bottom: 5px;">&nbsp;&nbsp;A standard curve in a linear relationship can be obtained by calibration experiments.</br>
 +
By comparing and analyzing the fluorescence values and Absorbance value of the different test devices at 0 and 6 hours, we can draw the following conclusions: the negative control and the positive control showed significant differences in the 6h fluorescence measurement results. Among the six different test equipment, Device 4 has the strongest fluorescence, while Device 3 has the lowest fluorescence. Compared with the relation between absorbance and fluorescence value, we can't get the relationship, and we need to improve on the control variables during the experiment.
 +
</p>
  
 +
            <h2 >Discussion</h2>
 +
            <p style="margin-bottom: 5px;">&nbsp;&nbsp;&nbsp;&nbsp;During the experiment, we encountered two problems.</br>
 +
&nbsp;&nbsp;1. In the cell measurement experiment, we need to dilute the cultures further to a target Abs600 of 0.02, but there are errors with in the actual operation, can not be accurately diluted to 0.02. So we hope that the protocol can provide acceptable error range.</br>
 +
&nbsp;&nbsp;2. In the second calibration, the particle standard curve log graph is not a straight line. We guess that it is due to pipetting error or the time to add the sample is too long.
  
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  <h3 style="text-align: left;">EurAsian Meetup</h3>
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  <img src="https://static.igem.org/mediawiki/2018/b/bc/T--FJNU-China--pick.png" style="width: 45%;">  <img src="https://static.igem.org/mediawiki/2018/b/bc/T--FJNU-China--pick.png" style="width: 45%;"> 
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  <p>As we know, the purpose of modeling is mainly to provide guidance and solve problems for the team experiment. So, before we designed the model, we sorted out some of the problems the team was facing. Based on these three problems,.Because The effect of PLA concentration on the quantity Staphylococcus epidermidis.Vestibulum quis quam ut magna consequat faucibus. Pellentesque eget nisi a mi suscipit tincidunt. Ut tempus dictum risus. Pellentesque viverra sagittis quam at mattis. Suspendisse potenti. Aliquam sit amet gravida nibh, facilisis gravida odio. Phasellus auctor velit at lacus blandit, commodo iaculis justo viverra. Etiam vitae est arcu.</p>
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    <h3 style="text-align: left;">5th CCiC</h3>
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  <img src="https://static.igem.org/mediawiki/2018/b/bc/T--FJNU-China--pick.png" style="width: 45%;">  <img src="https://static.igem.org/mediawiki/2018/b/bc/T--FJNU-China--pick.png" style="width: 45%;"> 
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  <p>As we know, the purpose of modeling is mainly to provide guidance and solve problems for the team experiment. So, before we designed the model, we sorted out some of the problems the team was facing. Based on these three problems,.Because The effect of PLA concentration on the quantity Staphylococcus epidermidis.Vestibulum quis quam ut magna consequat faucibus. Pellentesque eget nisi a mi suscipit tincidunt. Ut tempus dictum risus. Pellentesque viverra sagittis quam at mattis. Suspendisse potenti. Aliquam sit amet gravida nibh, facilisis gravida odio. Phasellus auctor velit at lacus blandit, commodo iaculis justo viverra. Etiam vitae est arcu.</p>
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            <h3>Reference</h3>
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            <p style="margin-bottom: 5px;font-size:15px;">1.Beal J, Haddock-Angelli T, Gershater M, de Mora K, Lizarazo M, Hollenhorst J, et al. (2016) Reproducibility of Fluorescent Expression from Engineered Biological Constructs in E. coli. PLoS ONE 11(3): e0150182. </br>
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2.https://2018.igem.org/Measurement/InterLab</br>
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3.http://parts.igem.org/Part:BBa_J61002
  
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Revision as of 09:59, 16 October 2018

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JUDGING FORM ⇗

Description

Overview

  Reliable and repeatable measurement is a key component to all engineering disciplines. However, the number of cells in the sample is a variability in measurements. The goal of the iGEM InterLab Study is to identify and correct the sources of systematic variability in synthetic biology measurements. This year, our team take part in the fifth InterLab study, using normalizing to absolute cell count or CFUs instead of OD to reduce variability of fluorescence measurements.

Materials

Strain used
Escherichia coli  DH5α

Plasmid used
Negative control: BBa_R0040 Plate 7 Well 2D
Positive control: BBa_I20270 Plate 7 Well 2B
Test Device 1: BBa_J364000 Plate 7 Well 2F
Test Device 2: BBa_J364001 Plate 7 Well 2H
Test Device 3: BBa_J364002 Plate 7 Well 2J
Test Device 4: BBa_J364007 Plate 7 Well 2L
Test Device 5: BBa_J364008 Plate 7 Well 2N
Test Device 6: BBa_J364009 Plate 7 Well 2P

Machine
Molecular Devices SpectraMax i3x

Methods

  We followed this protocol to do the Interlab Study.
  When we completed three of the calibration measurements, performing the cell measurements. Used the same plates, volumes and settings that we used in calibration protocol. We transformed E.coli DH5α competent cells with the 8 plasmids and picked 2 colonies from each of plates into 5 mL LB medium + Chloramphenicol. After culturing the cells overnight at 37°C and 220 rpm, we used plate reader to measure the Abs600 and fluorescence of samples at 0, 6 hours.

Result

Calibration
Calibration 1: OD600 reference point


Calibration 2: Particle Standard Curve

table1
figue34

Cell measurement

figue56

Analyse

  A standard curve in a linear relationship can be obtained by calibration experiments.
By comparing and analyzing the fluorescence values and Absorbance value of the different test devices at 0 and 6 hours, we can draw the following conclusions: the negative control and the positive control showed significant differences in the 6h fluorescence measurement results. Among the six different test equipment, Device 4 has the strongest fluorescence, while Device 3 has the lowest fluorescence. Compared with the relation between absorbance and fluorescence value, we can't get the relationship, and we need to improve on the control variables during the experiment.

Discussion

    During the experiment, we encountered two problems.
  1. In the cell measurement experiment, we need to dilute the cultures further to a target Abs600 of 0.02, but there are errors with in the actual operation, can not be accurately diluted to 0.02. So we hope that the protocol can provide acceptable error range.
  2. In the second calibration, the particle standard curve log graph is not a straight line. We guess that it is due to pipetting error or the time to add the sample is too long.

Reference

1.Beal J, Haddock-Angelli T, Gershater M, de Mora K, Lizarazo M, Hollenhorst J, et al. (2016) Reproducibility of Fluorescent Expression from Engineered Biological Constructs in E. coli. PLoS ONE 11(3): e0150182.
2.https://2018.igem.org/Measurement/InterLab
3.http://parts.igem.org/Part:BBa_J61002

 

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Attribution