Difference between revisions of "Team:Harvard/InterLab"

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         <h5>Materials</h5>
 
         <h5>Materials</h5>
 
         <ul>
 
         <ul>
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           <li>1 mL LUDOX</li>
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           <li></li>
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          <li>H2O</li>
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          <li>96 well plate</li>
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         </ul>
 
         </ul>
 
         <h5>Methods</h5>
 
         <h5>Methods</h5>
 
         <ol>
 
         <ol>
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           <li>100 uL of LUDOX-S40 from the InterLab Measurement Kit added into wells A1, B1, C1, and D1</li>
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           <li>Induce expression of curli by adding 1M IPTG to liquid culture, to a final concentration of 250 µM. For example, if you have 3 mL culture, add 0.75 µL of 1M IPTG solution.
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           <li>100 uL of dH2O added to wells A2, B2, C2, D2</li>
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          </li>
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           <li>Absorbance at 600 nm taken for all samples in setup described above</li>
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          <li>Incubate liquid culture overnight for optimal curli expression.</li>
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           <li>Data used to obtain correction factor</li>
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          <li>Make 0.015% Congo Red solution by diluting 1% congo red solution. For example, 150 µl of 1% congo red solution in 10 ml DI water.</li>
 +
          <li>Remove the culture from the 25C incubator. Add 1 ml of the culture into a cleaned 2ml Eppendorf tube.</li>
 +
          <li>Pellet the cells by centrifuge at 6800 g (8000 rpm) at room temperature for 10 minutes.</li>
 +
          <li>Remove the supernatant by decanting, and using a pipette to siphon off as much of  the supernatant without disturbing the pellet.</li>
 +
           <li>Resuspend cells in 1 mL of PBS gently by pipetting up and down with a pipette.</li>
 +
          <li>Add 100 µl of 0.015% congo red solution to the tube. Mix gently by pipetting. For the control tube, add the congo red solution to 1 ml of pure PBS.</li>
 +
           <li>Leave the solution at room temperature for 10 minutes</li>
 +
           <li>Pellet by centrifuging at 14,000 rpm at room temperature for 10 minutes.</li>
 +
          <li>Add 150 µl of the supernatant to a well of 96-well plate along with pure PBS, and control PBS-Congo Red.</li>
 +
          <li>Using a plate reader, measure absorbance at wavelength 490 nm. </li>
 
         </ol>
 
         </ol>
 +
      <p>Protocol Courtesy of Bom Praveschotinunt of Wyss Institute, Joshi Lab</p>
 
       </div>
 
       </div>
 
     </div>
 
     </div>

Revision as of 03:20, 30 October 2017

Harvard OPTI POLY

InterLab

Overview


Difficulty in taking reliable and reproducible measurements remains a key obstacle in establishing synthetic biology as an engineering discipline. The InterLab Study is part of the Measurement Committee’s continuing effort to develop a standard measurement procedure for green fluorescent protein (GFP). Despite being one of the most commonly used markers in synthetic biology, labs often resort to making relative comparisons, which makes it difficult for labs to share and data and/or constructs.
For the fourth installment of the InterLab, iGEM hopes to establish a GFP measurement protocol that can be used to produce comparable GFP measurements on different plate readers. In addition, iGEM teams from around the world have tested RBS devices (BCDs) intended to increase the precision and reliability of gene expression. The parts used include six test devices (BBa_J364000, BBa_J364001, BBa_J364002, BBa_J364003, BBa_J364004, BBa_J364005), as well as a positive (BBa_I20270) and negative (BBa_R0040) control. All parts are located in the pSB1C3 plasmid and carry chloramphenicol resistance.

Methods and Materials

Plate Reader Setup

Plate Reader: Synergy H1

Abs OD600
  • Wavelengths: 600
  • Read Speed: Normal
  • Delay: 100 msec
Fluorescence
  • Excitation: 395
  • Emission: 509
  • Optics: Top
  • Gain: 75
  • Light Source: Xenon Flash
  • Lamp Energy: High
  • Read Speed: Normal
  • Delay: 100 msec
  • Read Height: 7 mm

OD600 Reference Point

Materials
Methods
  1. Induce expression of curli by adding 1M IPTG to liquid culture, to a final concentration of 250 µM. For example, if you have 3 mL culture, add 0.75 µL of 1M IPTG solution.
  2. Incubate liquid culture overnight for optimal curli expression.
  3. Make 0.015% Congo Red solution by diluting 1% congo red solution. For example, 150 µl of 1% congo red solution in 10 ml DI water.
  4. Remove the culture from the 25C incubator. Add 1 ml of the culture into a cleaned 2ml Eppendorf tube.
  5. Pellet the cells by centrifuge at 6800 g (8000 rpm) at room temperature for 10 minutes.
  6. Remove the supernatant by decanting, and using a pipette to siphon off as much of the supernatant without disturbing the pellet.
  7. Resuspend cells in 1 mL of PBS gently by pipetting up and down with a pipette.
  8. Add 100 µl of 0.015% congo red solution to the tube. Mix gently by pipetting. For the control tube, add the congo red solution to 1 ml of pure PBS.
  9. Leave the solution at room temperature for 10 minutes
  10. Pellet by centrifuging at 14,000 rpm at room temperature for 10 minutes.
  11. Add 150 µl of the supernatant to a well of 96-well plate along with pure PBS, and control PBS-Congo Red.
  12. Using a plate reader, measure absorbance at wavelength 490 nm.

Protocol Courtesy of Bom Praveschotinunt of Wyss Institute, Joshi Lab

Protocol Fluorescein Fluorescence Standard Curve

Materials
  • Fluorescein
  • 10 mL 1xPBS (phosphate buffered saline)
  • 96 well plate
Methods
  1. Fluorescein stock tube from InterLab Measurement Kit spun down to make sure pellet is at bottom of tube
  2. 2x fluorescein stock solution (100 uM) prepared by resuspending fluorescein in 1 mL of 1xPBS
  3. 500 uL of 2x fluorescein stock solution diluted with 500 uL of 1xPBS to make 1mL of 50 uM (1x) fluorescein solution
  4. Serial dilutions of fluorescein:
    1. 100 uL of PBS added into wells A2, B2, C2, D2... A12, B12
    2. 200 uL of fluorescein 1x stock solution added to A1, B1, C1, D1
    3. 100 uL fluorescein stock solution transferred from A1 into A2
    4. A2 mixed by pipetting up and down, then 100 uL transferred to A3
    5. A3 mixed by pipetting up and down, then 100 uL transferred to A4
    6. A4 mixed by pipetting up and down, then 100 uL transferred to A5
    7. A5 mixed by pipetting up and down, then 100 uL transferred to A6
    8. A6 mixed by pipetting up and down, then 100 uL transferred to A7
    9. A8 mixed by pipetting up and down, then 100 uL transferred to A9
    10. A9 mixed by pipetting up and down, then 100 uL transferred to A10
    11. A10 mixed by pipetting up and down, then 100 uL transferred to A11
    12. A11 mixed by pipetting up and down, then 100 uL transferred to liquid waste
  5. Step 4 repeated for rows B-D
  6. Fluorescence for all samples measured using setup described above

Cell Measurements

Materials
  • Competent Cells (Escherichia coli strain DH5 alpha
  • LB media
  • Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH, working stock 25 ug/mL)
  • 50 mL Falcon tubes covered in foil
  • Incubator at 37ºC
  • Devices from InterLab Measurement Kit
    • Positive Control
    • Negative Control
    • Test Device 1: J23101 + I13504
    • Test Device 2: J23106 + I13504
    • Test Device 3: J23117 + I13504
    • Test Device 4: J23101.BCD2.E0040.B0015
    • Test Device 5: J23106.BCD2.E0040.B0015
    • Test Device 6: J23117.BCD2.E0040.B0015
Methods
  1. Each of the plasmids listed above were located on Plate 7 of the distribution kit and resuspended in 10 uL of dH2O
  2. Each device was transformed into 10 uL of competent cells (see here for the full transformation protocol)
  3. Transformations were spot plated onto an agar plate with chloramphenicol and grown overnight(see here for the full spot plating protocol)
  4. On Day 2, 2 colonies were picked from each plate and inoculated in 10 mL of LB medium + Chloramphenicol in 50 mL Falcon tubes covered with foil. These cultures were grown overnight in an incubator at 37ºC and 220 rpm.
  5. On Day 3, 100 uL of each culture was pipetted into a 96 well plate for an OD600 reading, which was then used for the Dilution Calculations in the Excel sheet provided by iGEM
  6. Each culture was diluted to a target OD of 0.02 following the preloading culture and media volumes calculated by the Dilution excel sheet (see OD Readings and Dilutions Calculations in the Results section below) in 12 mL of LB media + chloramphenicol in 50 mL falcon tubes wrapped in foil
  7. Cultures were placed in incubator at 37ºC and 220 rpm
  8. 4 replicates of 100 uL samples were taken from each culture at 0, 2, 4, and 6 hours of incubation and placed in a 96 well plate for OD and fluorescence measurements using the setup described above

Results

OD600 Calibration

OD Calibration

Fluorescein Standard Curve

Fluorescein Standard Curve

Dilution Calculations

Sample Abs600 Reading Volume of Preloading Culture (mL) Volume of Preloading Media (mL)
Positive control (1) 1.011 0.205 9.795
Positive control (2) 2.261 0.0900 9.910
Negative control 1.3 0.158 9.841
Negative control (2) 0.734 0.287 9.713
Device 1 (1) 0.443 1.493 8.507
Device 1 (2) 0.138 1.980 8.020
Device 2 (1) 0.425 2.515 7.485
Device 2 (2) 0.136 2.020 7.980
Device 3 (1) 0.507 0.426 9.574
Device 3 (2) 0.543 0.395 9.604
Device 4 (1) 0.306 0.743 9.257
Device 4 (2) 0.409 0.538 9.462
Device 5 (1) 0.195 0.266 9.734
Device 5 (2) 0.494 0.438 9.562
Device 6 (1) 1.597 0.357 9.643
Device 6 (2) 1.58 0.130 9.870
Media+chl 0.037

OD Readings

OD Readings

Averaged OD measurements of the 4 replicates for each sample, taken at two hour intervals.

Fluorescence Readings

Fluorescence Readings

Averaged fluorescence measurements of the 4 replicates for each sample, taken at two hour intervals.