Difference between revisions of "Team:Newcastle/InterLab"

 
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<h1 id="teams">Interlab</h1>
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<h2>Interlab</h2>
 
<p></p>
 
<p></p>
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<h2 id="Introduction">Introduction</h2>
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<h3 id="Introduction">Introduction</h3>
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<p>InterLab study 2016 is the largest interlaboratory study in synthetic biology. It aims to standardize the measurement of fluorescence in labs around the world by testing replicability of data. This time it is done by following the same protocols designed for a plate reader and a flow cytometer that will produce data in same units everywhere so it can be compared and analysed. Team Newcastle decided to test both protocols to measure the fluorescence.</p>
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<p>InterLab 2016 is the third, and largest, interlaboratory study ever conducted in synthetic biology. Data from the two previous InterLab studies is summarised in a paper by <a href="http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0150182">Beal et al. 2016</a>. The study aims to standardize the measurement of fluorescence in labs around the world by testing replicability of data. This time it is done by following the same protocols designed for a plate reader and a flow cytometer that will produce data in same units everywhere so it can be compared and analysed. Team Newcastle decided to test both protocols to measure the fluorescence.</p>
 
<h3 id="InterLab Measurement Kit">InterLab Measurement Kit</h3>
 
<h3 id="InterLab Measurement Kit">InterLab Measurement Kit</h3>
 
<p>This year teams were provided with the InterLab Measurement Kit which contained 7 tubes (5 with plasmid DNA and 2 with standard reagents):</p>
 
<p>This year teams were provided with the InterLab Measurement Kit which contained 7 tubes (5 with plasmid DNA and 2 with standard reagents):</p>
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<p>• Plasmid DNA (100 pg/uL in 10uL of Buffer EB)
 
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o Test Device 1: J23101.B0034.E0040.B0015 in pSB1C3
 
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o Test Device 2: J23106.B0034.E0040.B0015 in pSB1C3
 
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o Test Device 3: J23117.B0034.E0040.B0015 in pSB1C3
 
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o Positive Control Device: I20270 in pSB1C3
 
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o Negative Control Device: R0040 in pSB1C3
 
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• FITC Standard: one tube with dried down FITC for creating a FITC standard
 
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• LUDOX: one tube with 30% colloidal silica suspended in 1mL of water
 
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</p>
 
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<h4 id="Protocols">Protocols</h4>
 
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<p>E. cloni® 10G Chemically Competent Cells were used for the transformation of bacteria. The protocol followed can be found here: http://www.lucigen.com/docs/manuals/MA010-Ecloni-10G-Chem-Comp.pdf</p>
 
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<p>Transformed cells were plated on Petri dishes and grown in liquid cultures. They were prepared by following an official 2016 iGEM InterLab Measurement Study Plate Reader protocol and growing cultures in 10 ml LB and 10µl of 1000x chloramphenicol mixed medium.</p>
 
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<h5 id="2016 iGEM InterLab Measurement Study Plate Reader Protocol">2016 iGEM InterLab Measurement Study Plate Reader Protocol</h5>
 
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<p>First, fluorescence was measured with FLUOstar OMEGA Microplate Reader provided by BMG Labtech while following updated 2016 iGEM InterLab Measurement Study Plate Reader Protocol that can be found here: https://static.igem.org/mediawiki/2016/c/c5/InterLab_iGEM2016_Plate_Reader_Protocol_Updated_July.pdf</p>
 
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<p>While following cell measurement section of the protocol above, the target OD600 achieved after the first measurement with (insert brand) spectrophotometer and dilution according to the calculations in the sheet provided was 0.03 in all of the samples. The data which was obtained from the plate reader measurements can be found below.</p>
 
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<h6 id="2016 iGEM InterLab Measurement Study Flow Cytometry Protocol">2016 iGEM InterLab Measurement Study Flow Cytometry Protocol</h6>
 
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<p>2016 iGEM InterLab Measurement Study Flow Cytometry Protocol (https://2016.igem.org/Tracks/Measurement/Interlab_study/Flow_Cytometry_Overview) was followed to measure fluorescence by flow cytometry. FACSCanto II flow cytometer configured for measurement of GFP and SpheroTech Rainbow Calibration Particles RCP-30-5A were used as recommended in the protocol. The team were guided all the way by the members of staff working at the Flow Cytometry Core Facility of the Newcastle University to make sure that the machine was used properly and measurements obtained are right.</p>
 
−
<p>However, a few limitations might have affected the results. First of all, the protocol page did not have clear instructions how to prepare the cultures so they were diluted 2/3 and then 1/100 times to be measured in the flow cytometer. It would be useful to have a full flow cytometry protocol to avoid differences in dilutions. Also, geometric mean could not be obtained for the negative control and devices that had no GFP expression due to them having a negative value. Thus, other type of data could be collected to get more accurate and consistent data.</p>
 
  
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<p><a href="https://2016.igem.org/Team:Exeter/Collaborations">Team Exeter</a> kindly helped us by conducting thermal conductivity experiments on LB and M9 media. These were important in allowing us to correctly model the temperature change caused by running an electric current through common bacterial growth media. They found that the conductivity of both <a href="">M9</a> and <a href="">LB</a> to be very similar to that of water. Whilst water has a thermal conductivity of 598.4 mW/Km (miliwatts per metre kelvin) at room temperature Exeter found that LB has a slightly higher specific heat capacity at 605 (+/- 20) mW/Km and M9 a slightly lower specific heat capacity of 570 (+/- 30) mW/Km.</p>
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<ul class="dot">
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<p>This data was incorporated into <a href="https://2016.igem.org/Team:Newcastle/Notebook/Lab/Modelling/">our modelling</a> and helped us identify which media to use in our later conductivity experiments. It also gave us some useful pointers for exploring other fluids with different specific heat capacities. Thanks Exeter!</p>
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<li><p>Plasmid DNA (100 pg/uL in 10 uL of Buffer EB)</p> </li>
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<h2 id="Westminster meet up">Westminster meet up</h2>
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<li><p>Test Device 1: J23101.B0034.E0040.B0015 in pSB1C3</p> </li>
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<p> On the 17-18th of August a half of Newcastle University iGEM 2016 team went to UK iGEM teams meet up in University of Westminster where we had to display our poster and make the presentation.</p>  
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<li><p>Test Device 2: J23106.B0034.E0040.B0015 in pSB1C3</p> </li>
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<p>The presentation went well, but a few improvements could be made. First of all, the clear structure of the presentation that had separate sections for introduction, microfluidics and breadboard, genetics and human practices worked well. We will try to make our next presentation very structural as well. Also, a breadboard design picture should be included in order to help people visualize how our final product looks like. Our human practices part looked very integrative and different compared with other teams and now we should continue and actually develop a human practices simulator. Finally, chronological order in some parts of the presentation such as human practices were easy for listeners to understand and follow so we are planning to use this approach in other parts of our presentation. </p>
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<li><p>Test Device 3: J23117.B0034.E0040.B0015 in pSB1C3</p> </li>
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<p>The poster was received positively as well. However, when comparing ours with the ones of other teams we have indicated a few areas which could be improved. First of all, our poster could have contained less text and maybe bullet points to make it more attractive. By reducing the amount of text in the poster we are also able to make the heading and text larger. Finally, we would like to make the sections of our final poster more separate than before. Even though this poster had a better separation than the previous one, the components description were a little bit scattered and thus harder to follow.</p>
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<li><p>Positive Control Device: I20270 in pSB1C3 </p> </li>
 +
<li><p>Negative Control Device: R0040 in pSB1C3 </p> </li>
 +
<li><p>FITC Standard: one tube with dried down FITC for creating a FITC standard </p> </li>
 +
<li><p>LUDOX: one tube with 30% colloidal silica suspended in 1 mL of water </p> </li>
 +
</ul>
 +
 
 +
<h3 id="Protocols">Protocols</h3>
 +
<p>E. cloni® 10G Chemically Competent Cells were used for the transformation of bacteria. You can find the protocol we use for our transformations <a href="https://2016.igem.org/Team:Newcastle/Protocols#ecloni-transformation"> in our protocol library</a>.</p>
 +
<p>Transformed cells were plated on Petri dishes and grown in liquid cultures with appropriate antibiotic. They were prepared by following an official 2016 iGEM InterLab Measurement Study Plate Reader protocol and growing cultures in 10 ml LB and 10 µl of 1000x chloramphenicol mixed medium.</p>
 +
<h4 id="2016 iGEM InterLab Measurement Study Plate Reader Protocol">2016 iGEM InterLab Measurement Study Plate Reader Protocol</h4>
 +
<p>First, fluorescence was measured with FLUOstar OMEGA Microplate Reader provided by BMG Labtech while following updated 2016 iGEM InterLab Measurement Study Plate Reader Protocol that can be found <a href="https://static.igem.org/mediawiki/2016/c/c5/InterLab_iGEM2016_Plate_Reader_Protocol_Updated_July.pdf">here</a>.</p>
 +
<p>While following cell measurement section of the protocol above, the target OD<sub>600</sub> achieved after the first measurement with an Eppendorf Biophotometer and dilution according to the calculations in the sheet provided was 0.03 in all of the samples. The data which was obtained from the plate reader measurements can be found below.</p>
 +
<h4 id="2016 iGEM InterLab Measurement Study Flow Cytometry Protocol">2016 iGEM InterLab Measurement Study Flow Cytometry Protocol</h4>
 +
 
 +
<p><a href="https://2016.igem.org/Tracks/Measurement/Interlab_study/Flow_Cytometry_Overview">2016 iGEM InterLab Measurement Study Flow Cytometry Protocol</a> was followed to measure fluorescence by flow cytometry. FACSCanto II flow cytometer configured for measurement of GFP and SpheroTech Rainbow Calibration Particles RCP-30-5A were used as recommended in the protocol. The team were guided all the way by the members of staff working at the Flow Cytometry Core Facility, Newcastle University to make sure that the machine was used properly and measurements obtained are right.</p>
 +
<p>However, a few limitations might have affected the results. First of all, the protocol page did not have clear instructions how to prepare the cultures so they were diluted 2:3, and then 1:100 times to be measured in the flow cytometer. It would be useful to have a full flow cytometry protocol to avoid differences in dilutions. Also, the geometric mean could not be obtained for the negative control and devices that had no GFP expression due to them having a negative value. Thus, other types of data could be collected to get more accurate and consistent data.</p>
 +
 
 +
 
 +
<p><h3>Results</h3></p>
 +
<p>Both Plate Reader and Flow Cytometry results indicate that Device 1 is the closest to positive control which means that it has the strongest promoter and the best RBS. The other two devices produce results which are close to negative control data. This indicates that they have very week or non-functioning promoters and RBS.  
 +
 
 +
<p>Our results can be found <a href="https://2016.igem.org/Team:Newcastle/InterLab_Results">here</a></p>
 
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Latest revision as of 22:50, 19 October 2016


Interlab

Introduction

InterLab 2016 is the third, and largest, interlaboratory study ever conducted in synthetic biology. Data from the two previous InterLab studies is summarised in a paper by Beal et al. 2016. The study aims to standardize the measurement of fluorescence in labs around the world by testing replicability of data. This time it is done by following the same protocols designed for a plate reader and a flow cytometer that will produce data in same units everywhere so it can be compared and analysed. Team Newcastle decided to test both protocols to measure the fluorescence.

InterLab Measurement Kit

This year teams were provided with the InterLab Measurement Kit which contained 7 tubes (5 with plasmid DNA and 2 with standard reagents):

  • Plasmid DNA (100 pg/uL in 10 uL of Buffer EB)

  • Test Device 1: J23101.B0034.E0040.B0015 in pSB1C3

  • Test Device 2: J23106.B0034.E0040.B0015 in pSB1C3

  • Test Device 3: J23117.B0034.E0040.B0015 in pSB1C3

  • Positive Control Device: I20270 in pSB1C3

  • Negative Control Device: R0040 in pSB1C3

  • FITC Standard: one tube with dried down FITC for creating a FITC standard

  • LUDOX: one tube with 30% colloidal silica suspended in 1 mL of water

Protocols

E. cloni® 10G Chemically Competent Cells were used for the transformation of bacteria. You can find the protocol we use for our transformations in our protocol library.

Transformed cells were plated on Petri dishes and grown in liquid cultures with appropriate antibiotic. They were prepared by following an official 2016 iGEM InterLab Measurement Study Plate Reader protocol and growing cultures in 10 ml LB and 10 µl of 1000x chloramphenicol mixed medium.

2016 iGEM InterLab Measurement Study Plate Reader Protocol

First, fluorescence was measured with FLUOstar OMEGA Microplate Reader provided by BMG Labtech while following updated 2016 iGEM InterLab Measurement Study Plate Reader Protocol that can be found here.

While following cell measurement section of the protocol above, the target OD600 achieved after the first measurement with an Eppendorf Biophotometer and dilution according to the calculations in the sheet provided was 0.03 in all of the samples. The data which was obtained from the plate reader measurements can be found below.

2016 iGEM InterLab Measurement Study Flow Cytometry Protocol

2016 iGEM InterLab Measurement Study Flow Cytometry Protocol was followed to measure fluorescence by flow cytometry. FACSCanto II flow cytometer configured for measurement of GFP and SpheroTech Rainbow Calibration Particles RCP-30-5A were used as recommended in the protocol. The team were guided all the way by the members of staff working at the Flow Cytometry Core Facility, Newcastle University to make sure that the machine was used properly and measurements obtained are right.

However, a few limitations might have affected the results. First of all, the protocol page did not have clear instructions how to prepare the cultures so they were diluted 2:3, and then 1:100 times to be measured in the flow cytometer. It would be useful to have a full flow cytometry protocol to avoid differences in dilutions. Also, the geometric mean could not be obtained for the negative control and devices that had no GFP expression due to them having a negative value. Thus, other types of data could be collected to get more accurate and consistent data.

Results

Both Plate Reader and Flow Cytometry results indicate that Device 1 is the closest to positive control which means that it has the strongest promoter and the best RBS. The other two devices produce results which are close to negative control data. This indicates that they have very week or non-functioning promoters and RBS.

Our results can be found here