Difference between revisions of "Team:McMasterU/InterLab"

(Prototype team page)
 
Line 19: Line 19:
 
</p>
 
</p>
 
</div>
 
</div>
 +
 +
<h1>Overview</h1>
 +
<p>The Interlab Study, developed by the Measurement Committee of iGEM as a way to standardize measurements for green fluorescent protein (GFP), tackles the foundational synthetic biology problem of a lack of reliable measurements for experiments. With an international standard set down, scientists can go beyond utilizing relativity comparisons for results, to conduct quantitative reproducibility studies across labs. This Interlab experiment, carried out by McMaster iGEM with plate readers, aims to produce comparable GFP measurements as a contribution for the international standardization of GFP values. The parts we used were BBA_J364000 (1), BBa_J364001 (2), BBa_J364002 (3), BBa_J36003 (4), BBa_J364004 (5), BBa_J364005 (6), and positive control BBa_120270  with negative control BBa_R0040. All parts were located in pSB1C3 plasmid and were modified for chloramphenicol resistance.
 +
<br><br>
 +
<h1>Methods and Materials</h1>
 +
<h2>Plate Reader</h2>
 +
<p>Plate Reader: TECAN Infinite M1000
 +
Plate: Corning 96 Flat Bottom black, clear bottom Polystyrol (COR96fb clear bottom)
 +
<br>
 +
<strong>Abs OD600</strong>
 +
<ul>Wavelengths: 600 nm</ul>
 +
<ul>Number of Flashes: 20</ul>
 +
<ul>Settle Time: 0 ms</ul>
 +
<ul>Temperature: 26.1 degrees Celsius</ul>
 +
<br>
 +
<strong>Fluorescence</strong>
 +
<ul>Excitation:485 nm</ul>
 +
<ul>Emission: 535 nm</ul>
 +
<ul>Excitation Bandwidth: 5 nm</ul>
 +
<ul>Emission Bandwidth: 5 nm</ul>
 +
<ul>Optics: Bottom Reading</ul>
 +
<ul>Gain: Optimal (automatic)</ul>
 +
<ul>Number of Flashes: 50</ul>
 +
<ul>Flash Frequency: 400 Hz</ul>
 +
<ul>Integration Time: 20 us</ul>
 +
<ul>Lag Time: 0 us</ul>
 +
<ul>Settle Time: 0 ms</ul>
 +
<ul>Part of Plate: A1-H10</ul>
 +
<ul>Temperature: 26.8 degrees Celsius</ul>
 +
<br><br>
 +
 +
<h2>OD600 Reference Point</h2>
 +
<strong>Materials</strong>
 +
<ul> 1 mL LUDOX (provided for by iGEM Headquarters)</ul>
 +
<ul>Distilled H2O</ul>
 +
<ul>96 well plate, Corning 96 Flat Bottom black, clear bottom Polystyrol (COR96fb clear bottom)</ul>
 +
<br>
 +
<strong>Methods</strong>
 +
<ol>
 +
<li>Add 100 uL of LUDOX into A1, B1, C1, and D1 wells</li>
 +
<li>Add 1 mL LUDOX into a separate cuvette</li>
 +
<li>Add 100 uL of dH2O into wells A2, B2, C2, D2 wells</li>
 +
<li>Add 1 mL dH2O into a separate cuvette</li>
 +
<li>Image using plate reader (TECAN Infinite M1000)</li>
 +
</ol>
 +
<br><br>
 +
 +
<h2>Protocol of Fluorescein Fluorescence Standard Curve</h2>
 +
<strong>Materials:</strong>
 +
<ul>
 +
<li>Fluorescein</li>
 +
<li>10 mL 1x PBS (phosphate buffered saline)</li>
 +
<li>96 well plate, Corning 96 Flat Bottom black, clear bottom Polystyrol (COR96fb clear bottom)</li>
 +
</ul>
 +
<br>
 +
<strong>Methods:</strong>
 +
<ol>
 +
<li>Spin down fluorescence stock tube until pellet is at bottom of tube</li>
 +
<li>Resuspend 100 uM stock Fluorescein in 1 mL of 1x PBS to obtain 2x fluorescein stock solution.</li>
 +
<li>Ensure that Fluorescein is properly dissolved.</li>
 +
<li>Dilute 2x fluorescin stock solution with 1x PBS to make a 1x fluorescein solution. Resulting concentration of fluorescein stock solution should be 50 uM (otherwise known as 500 uL of 2x fluoresceine in 500 uL of 1x PBS that makes 1 mL of 50 uM of 1x fluorescein solution).</li>
 +
<li>Prepare for serial dilutions across columns 1 to 11. Ensure column 12 has PBS buffer only.
 +
<ul>
 +
<li>Pipette 200 uL of 1x fluorescein stock into wells A1, B1, C1, D1.</li>
 +
<li>Pipette 100 uL of PBS in wells A2, B2, C2, D2 ... A12, B12.</li> 
 +
<li>Transfer 100 uL of fluorescein stock solution from A1 to A2.</li> 
 +
<li>Mix A2 by pipetting up and down 3x and transfer 100 uL into A3.</li> 
 +
<li>Mix A3 by pipetting up and down 3x and transfer 100 uL into A4.</li> 
 +
<li>Repeat 5c to 5e for A4, A5, A6.... to A11.</li> 
 +
<li>Mix A11 by pipetting up and down 3x and then transfer 100 uL into liquid waste. Ensure that A12 is PBS only</li>
 +
</ul>
 +
</li>
 +
<li>Repeat step 5 for columns B, C, and D.</li>
 +
<li>Measure fluorescence of all samples in the plate reader (settings listed above in Plate Reader Setup menu).</li> 
 +
<li>Record data in Fluorescein Standard Curve excel sheet and graph.</li>
 +
</ol>
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
  
  

Revision as of 06:22, 27 October 2017

HOME
TEAM
PROJECT
Description
Design
Experiments
Notebook
InterLab
Contribution
Model
Results
Demonstrate
Improve
Attributions
PARTS
Parts
Basic Parts
Composite Parts
Part Collection
SAFETY
HUMAN PRACTICES
Silver HP
Integrated and Gold
Public Engagement
AWARDS
Applied Design
Entrepreneurship
Hardware
Measurement
Model
Plant
Software
JUDGING FORM

McMasterU

★ ALERT!

This page is used by the judges to evaluate your team for the medal criterion or award listed above.

Delete this box in order to be evaluated for this medal criterion and/or award. See more information at Instructions for Pages for awards.

InterLab

Bronze Medal Criterion #4

Standard Tracks: Participate in the Interlab Measurement Study and/or improve the characterization of 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 2017 part number range.

For teams participating in the InterLab study, all work must be shown on this page.

Overview

The Interlab Study, developed by the Measurement Committee of iGEM as a way to standardize measurements for green fluorescent protein (GFP), tackles the foundational synthetic biology problem of a lack of reliable measurements for experiments. With an international standard set down, scientists can go beyond utilizing relativity comparisons for results, to conduct quantitative reproducibility studies across labs. This Interlab experiment, carried out by McMaster iGEM with plate readers, aims to produce comparable GFP measurements as a contribution for the international standardization of GFP values. The parts we used were BBA_J364000 (1), BBa_J364001 (2), BBa_J364002 (3), BBa_J36003 (4), BBa_J364004 (5), BBa_J364005 (6), and positive control BBa_120270 with negative control BBa_R0040. All parts were located in pSB1C3 plasmid and were modified for chloramphenicol resistance.

Methods and Materials

Plate Reader

Plate Reader: TECAN Infinite M1000 Plate: Corning 96 Flat Bottom black, clear bottom Polystyrol (COR96fb clear bottom)
Abs OD600

    Wavelengths: 600 nm
    Number of Flashes: 20
    Settle Time: 0 ms
    Temperature: 26.1 degrees Celsius

Fluorescence
    Excitation:485 nm
    Emission: 535 nm
    Excitation Bandwidth: 5 nm
    Emission Bandwidth: 5 nm
    Optics: Bottom Reading
    Gain: Optimal (automatic)
    Number of Flashes: 50
    Flash Frequency: 400 Hz
    Integration Time: 20 us
    Lag Time: 0 us
    Settle Time: 0 ms
    Part of Plate: A1-H10
    Temperature: 26.8 degrees Celsius


OD600 Reference Point

Materials
    1 mL LUDOX (provided for by iGEM Headquarters)
    Distilled H2O
    96 well plate, Corning 96 Flat Bottom black, clear bottom Polystyrol (COR96fb clear bottom)

Methods
  1. Add 100 uL of LUDOX into A1, B1, C1, and D1 wells
  2. Add 1 mL LUDOX into a separate cuvette
  3. Add 100 uL of dH2O into wells A2, B2, C2, D2 wells
  4. Add 1 mL dH2O into a separate cuvette
  5. Image using plate reader (TECAN Infinite M1000)


Protocol of Fluorescein Fluorescence Standard Curve

Materials:
  • Fluorescein
  • 10 mL 1x PBS (phosphate buffered saline)
  • 96 well plate, Corning 96 Flat Bottom black, clear bottom Polystyrol (COR96fb clear bottom)

Methods:
  1. Spin down fluorescence stock tube until pellet is at bottom of tube
  2. Resuspend 100 uM stock Fluorescein in 1 mL of 1x PBS to obtain 2x fluorescein stock solution.
  3. Ensure that Fluorescein is properly dissolved.
  4. Dilute 2x fluorescin stock solution with 1x PBS to make a 1x fluorescein solution. Resulting concentration of fluorescein stock solution should be 50 uM (otherwise known as 500 uL of 2x fluoresceine in 500 uL of 1x PBS that makes 1 mL of 50 uM of 1x fluorescein solution).
  5. Prepare for serial dilutions across columns 1 to 11. Ensure column 12 has PBS buffer only.
    • Pipette 200 uL of 1x fluorescein stock into wells A1, B1, C1, D1.
    • Pipette 100 uL of PBS in wells A2, B2, C2, D2 ... A12, B12.
    • Transfer 100 uL of fluorescein stock solution from A1 to A2.
    • Mix A2 by pipetting up and down 3x and transfer 100 uL into A3.
    • Mix A3 by pipetting up and down 3x and transfer 100 uL into A4.
    • Repeat 5c to 5e for A4, A5, A6.... to A11.
    • Mix A11 by pipetting up and down 3x and then transfer 100 uL into liquid waste. Ensure that A12 is PBS only
  6. Repeat step 5 for columns B, C, and D.
  7. Measure fluorescence of all samples in the plate reader (settings listed above in Plate Reader Setup menu).
  8. Record data in Fluorescein Standard Curve excel sheet and graph.