Difference between revisions of "Team:BioMarvel/InterLab"

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<h3>★  ALERT! </h3>
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<img class="menuIcon" src="https://static.igem.org/mediawiki/2018/c/c0/T--Biomarvel--interlab.jpg">
<p>This page is used by the judges to evaluate your team for the <a href="https://2018.igem.org/Judging/Medals">medal criterion</a> or <a href="https://2018.igem.org/Judging/Awards"> award listed below</a>. </p>
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<p> Delete this box in order to be evaluated for this medal criterion and/or award. See more information at <a href="https://2018.igem.org/Judging/Pages_for_Awards"> Instructions for Pages for awards</a>.</p>
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<p>The InterLab study is organized by a measurement committee of iGEM to establish better measurement techniques in the synthetic biology field.We submit our measurement results that cope with the absorbance and fluorescence related withE. coli transformed with a variety oftest devices. We checked 8 plasmids, containing 2 controls and 6 test devices, and measurements were made in a plate reader with a 96 well microplate. We measured the absorbance and fluorescence of our samples using a Perkin Elmer 1420 multilabel counter.</p>
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<h1>Calibration 1: OD <sub>600</sub> Reference point - LUDOX Protocol</h1>
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<p>We used LUDOX CL-X as a single point reference to obtain a conversion factor to transform absorbance (Abs <sub>600</sub>) data fromplate reader into acomparable OD <sub>600</sub> measurement as would be obtained in a spectrophotometer.</p>
 
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<img class="myimg" src="https://static.igem.org/mediawiki/2018/d/db/T--Biomarvel--inter1.png">
 
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<h4 style="text-align: center;">Fig 1. Measurement of Abs <sub>600</sub> and OD <sub>600</sub> for LUDOX CL-Xand water</h4>
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<h3>Bronze Medal Criterion #4</h3>
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<h1>Calibration 2: Particle Standard Curve - Microsphere Protocol</h1>
<p><b>Standard Tracks:</b> Participate in the Interlab Measurement Study and/or obtain new, high quality experimental characterization data for an existing BioBrick Part or Device and enter this information on that part's Main Page in the Registry. The part that you are characterizing must NOT be from a 2018 part number range.
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<p>We prepared a dilution series of silica microspheres and measure the Abs <sub>600</sub> in a plate reader. These microspheres are similar to cells in physical size and optical characteristics. This measurement was used to obtain a standardcurve of particle concentration, enabling to convert Abs <sub>600</sub> measurements to an estimatednumber of cells.</p>
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<img class="myimg" src="https://static.igem.org/mediawiki/2018/c/ce/T--Biomarvel--inter2.png">
For teams participating in the <a href="https://2018.igem.org/Measurement/InterLab">InterLab study</a>, all work must be shown on this page.  
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<h4 style="text-align: center;">Fig2. Calculated particle counts from measured Abs<sub>600</sub>and generation of a particle standard curve</h4>
 
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<h1>Calibration 3: Fluorescence standard curve - Fluorescein Protocol</h1>
 
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<p>We prepared a dilution series of fluorescein in four replicates and measure the fluorescence in a96 well plate in aplate reader. Measuring these in the plate readerenabled us togenerate astandard curve of fluorescence for fluorescein concentration.</p>
 
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<img class="myimg" src="https://static.igem.org/mediawiki/2018/c/ca/T--Biomarvel--inter3.png">
 
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<h4 style="text-align: center;">Fig 3.Correspondence between fluorescence and fluorescein concentration</h4>
 
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<h1>Cell measurement</h1>
 
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<p>We had two plates to read at the end of procedure and one plate for each time point: 0 and 6 hours. On each plate,we readboth fluorescence and absorbance.We used the generated particle standard and fluorescent curves to determine fluorescence per OD and fluorescence per particle values.</p>
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<img class="myimg" src="https://static.igem.org/mediawiki/2018/e/ef/T--Biomarvel--inter4.png">
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<h4 style="text-align: center;">Fig 4. Measurement of raw Abs600 and OD<sub>600</sub></h4>
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<img class="myimg" src="https://static.igem.org/mediawiki/2018/8/8c/T--Biomarvel--inter5.png">
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<h4 style="text-align: center;">Fig 5. Measurement of fluorescence per OD</h4>
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<img class="myimg" src="https://static.igem.org/mediawiki/2018/e/e0/T--Biomarvel--inter6.png">
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<h4 style="text-align: center;">Fig 6. Measurement of fluorescence per particle</h4>
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<img class="myimg" src="https://static.igem.org/mediawiki/2018/d/de/T--Biomarvel--inter7.png">
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<h4 style="text-align: center;">Fig 7. The number of colonies and CFU/mL value in a starting sample of OD<sub>600</sub> = 0.1</h4>
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<h1>Colony Forming Units per 0.1 OD<sub>600</sub><i>E. coli</i> cultures</h1>
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<p>For the CFU protocol, wecalculated colonies for our two Positive Control (BBa_I20270) cultures and our twoNegative Control (BBa_R0040) cultures.Our overnight cultureswere diluted to yield a 0.1 OD<sub>600</sub> using calculations described in iGEM 2018 InterLab Study Protocol.After a serial dilution for each overnight culture, we platedthe 8*10<sup>-3</sup>, 8*10<sup>-4</sup>, and 8*10<sup>-5</sup> dilutions on LB agar with triplication.With the assumption that one bacterial cell leads to one colony, we enumerated the number of colonies on each plate and multiplied the colony count by the final dilution factor to assess a CFU/mL value for a starting sample of OD<sub>600</sub>.</p>
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<img class="myimg" src="https://static.igem.org/mediawiki/2018/e/e2/T--Biomarvel--inter8.png">
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<h4 style="text-align: center;">Fig 8. Plated DH5α cells of serial dilutions for starting samples</h4>
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Revision as of 11:06, 8 October 2018

 

The InterLab study is organized by a measurement committee of iGEM to establish better measurement techniques in the synthetic biology field.We submit our measurement results that cope with the absorbance and fluorescence related withE. coli transformed with a variety oftest devices. We checked 8 plasmids, containing 2 controls and 6 test devices, and measurements were made in a plate reader with a 96 well microplate. We measured the absorbance and fluorescence of our samples using a Perkin Elmer 1420 multilabel counter.

Calibration 1: OD 600 Reference point - LUDOX Protocol

We used LUDOX CL-X as a single point reference to obtain a conversion factor to transform absorbance (Abs 600) data fromplate reader into acomparable OD 600 measurement as would be obtained in a spectrophotometer.

Fig 1. Measurement of Abs 600 and OD 600 for LUDOX CL-Xand water

Calibration 2: Particle Standard Curve - Microsphere Protocol

We prepared a dilution series of silica microspheres and measure the Abs 600 in a plate reader. These microspheres are similar to cells in physical size and optical characteristics. This measurement was used to obtain a standardcurve of particle concentration, enabling to convert Abs 600 measurements to an estimatednumber of cells.

Fig2. Calculated particle counts from measured Abs600and generation of a particle standard curve

Calibration 3: Fluorescence standard curve - Fluorescein Protocol

We prepared a dilution series of fluorescein in four replicates and measure the fluorescence in a96 well plate in aplate reader. Measuring these in the plate readerenabled us togenerate astandard curve of fluorescence for fluorescein concentration.

Fig 3.Correspondence between fluorescence and fluorescein concentration

Cell measurement

We had two plates to read at the end of procedure and one plate for each time point: 0 and 6 hours. On each plate,we readboth fluorescence and absorbance.We used the generated particle standard and fluorescent curves to determine fluorescence per OD and fluorescence per particle values.

Fig 4. Measurement of raw Abs600 and OD600

Fig 5. Measurement of fluorescence per OD

Fig 6. Measurement of fluorescence per particle

Fig 7. The number of colonies and CFU/mL value in a starting sample of OD600 = 0.1

Colony Forming Units per 0.1 OD600E. coli cultures

For the CFU protocol, wecalculated colonies for our two Positive Control (BBa_I20270) cultures and our twoNegative Control (BBa_R0040) cultures.Our overnight cultureswere diluted to yield a 0.1 OD600 using calculations described in iGEM 2018 InterLab Study Protocol.After a serial dilution for each overnight culture, we platedthe 8*10-3, 8*10-4, and 8*10-5 dilutions on LB agar with triplication.With the assumption that one bacterial cell leads to one colony, we enumerated the number of colonies on each plate and multiplied the colony count by the final dilution factor to assess a CFU/mL value for a starting sample of OD600.

Fig 8. Plated DH5α cells of serial dilutions for starting samples

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