Difference between revisions of "Team:UNITN-Trento/Test"

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<p>The confirmed devices were then transformed in different bacterial strains of E. Coli, NEB10β, NEB Express, and JM109. Each measurement was taken at the same optical density to allow a more precise comparison of the data. For each device we have 3 biological and 3 technical measurements for each used technique. We measured in vivo fluorescence emission in different ways using Tecan Infinite 200 PRO plate reader, Varian Cary Eclipse spectrofluorimeter, and BD FACSCanto FACS.</p>
 
<p>The confirmed devices were then transformed in different bacterial strains of E. Coli, NEB10β, NEB Express, and JM109. Each measurement was taken at the same optical density to allow a more precise comparison of the data. For each device we have 3 biological and 3 technical measurements for each used technique. We measured in vivo fluorescence emission in different ways using Tecan Infinite 200 PRO plate reader, Varian Cary Eclipse spectrofluorimeter, and BD FACSCanto FACS.</p>
 
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<p>The confirmed devices were then transformed in different bacterial strains of E. Coli, NEB10β, NEB Express, and JM109. Each measurement was taken at the same optical density to allow a more precise comparison of the data. For each device we have 3 biological and 3 technical measurements for each used technique. We measured in vivo fluorescence emission in different ways using Tecan Infinite 200 PRO plate reader, Varian Cary Eclipse spectrofluorimeter, and BD FACSCanto FACS.</p>
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<p>We also focused of transcription since the characterization is about promoters. To do so we we performed RT-qPCR using a BioRad CFX96 Touch™ Real-Time PCR Detection System. Additionally, we performed an in vitro characterization study, by measuring the fluorescence intensities of each device with a Cell Free <span class="i_enph italic">E. coli</span><span class="i_enph">S30</span> Extract System with a Circular DNA Real Time PCR.</p>
 
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Revision as of 14:35, 22 August 2015

Interlab Study

I have no idea what I'm doing!

  • Introduction & Achievements

  • Experimental Design

  • Experiments& Protocols

  • Final Discussion & Results

The Interlab Measurement Study

The characterization of standard parts has always been one of the main concerns in Synthetic Biology. For this very same reason, iGEM teams from all around the World were suggested to take part in the biggest Measurement Interlab Study ever conducted and the 2015 UNITN iGEM Team answered the call. The goal of this Second International Measurement Interlab Study is to assemble three different devices, each one containing a promoter with a screening plasmid intermediate and collect as many fluorescence data as possible. The three different promoters will differently affect the GFP production and thating plasmid intermediate and collect as iGEM teams are free to use any technique to measure their devices as long the obtained data are solid and reproducible.

Clear Data and Protocols

We have listed in the ”InterLab Study” page:

  • All devices measured for this study
  • All protocols developed and adopted
  • Sequencing data for all measurements devices

3 BioBrick devices

We used the three BioBrick devices listed in the ”Required Devices” section.

5 techniques

We used in-vivo and in-vitro techniques for measuring RNA and proteins levels:

  • Plate Reader
  • Spectrofluorimeter
  • FACS
  • RT-qPCR
  • Cell-Free Extract

Statistical Reliability

We have three biological replicates for each measurement, with positive and negative controls

Worksheet and Protocol

We have completed the InterLab Worksheet and the InterLab Protocol

Technical replicates

We have technical replicates for each sample

Experimental Design

We used the three mandatory devices for the measurement study:

The measurement devices were prepared by amplifying the reporter (BBa_I20270) by PCR. The amplified insert was then cut with XbaI and PstI and ligated into the plasmid containing the promoter previously cut with SpEI and PstI. All the devices were confirmed by restriction digestion as well as DNA sequencing.


In-vivo Measurements

The confirmed devices were then transformed in different bacterial strains of E. Coli, NEB10β, NEB Express, and JM109. Each measurement was taken at the same optical density to allow a more precise comparison of the data. For each device we have 3 biological and 3 technical measurements for each used technique. We measured in vivo fluorescence emission in different ways using Tecan Infinite 200 PRO plate reader, Varian Cary Eclipse spectrofluorimeter, and BD FACSCanto FACS.


In-vitro Measurements

We also focused of transcription since the characterization is about promoters. To do so we we performed RT-qPCR using a BioRad CFX96 Touch™ Real-Time PCR Detection System. Additionally, we performed an in vitro characterization study, by measuring the fluorescence intensities of each device with a Cell Free E. coliS30 Extract System with a Circular DNA Real Time PCR.