Team:Exeter/InterLab


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Cell Absorbance   Cell Measurements   Flow Cytometry   Colony Forming Units

What is InterLab?

The InterLab is the collaboration of iGEM teams to help identify and eliminate problems faced when sharing data between labs. Different instruments, such as plate readers, can give different results in different labs, despite following the same protocol. The question InterLab 2018 aims to answer is:

“Can we reduce lab-to-lab variability in fluorescence measurements by normalising to absolute cell count or colony-forming units (CFUs) instead of OD?”


Protocols: The InterLab study was divided into four categories; cell absorbance, cell measurements, flow cytometry, and colony forming units. The calibrations under cell absorbance involved a Tecan Infinite 200 plate reader.


Cell Absorbance:

Calibration 1: OD600 Reference Point (LUDOX Protocol)

    Why? To obtain a single reference point for converting absorbance (Abs600) into comparable OD600 measurement.

  1. 100 μL of LUDOX was pipetted into wells A1, B1, C1, and D1 of the 96 well plate.
  2. 100 μL of Molecular Biology water (ddH20) was pipetted into wells A2-D2.
  3. The absorbance at 600nm was measured for the plate, with the same settings for all future cell samples, in a Tecan Infinite M200.
  4. The data was recorded and imported into OD600 reference point, in the Excel sheet.


Calibration 2: Particle Standard Curve (Microsphere Protocol)

    Why? Allows us to construct a standard curve of particle concentration.

  1. 300μL Silica beads in a microsphere suspension were vortexed for approximately 30 seconds.
  2. 96μL of silicon beads were pipetted into a 1.5mL eppendorf tube directly after vortexing.
  3. 904μL of ddH20 was then added.
  4. This mixture was then vortexed for 10 seconds to make the Microsphere Stock Solution.
  5. 100 μL of water was added to wells A(2-12), B(2-12), C(2-12), and D(2-12) of a Corning 96-well flat black plate. Then 200μL of the Microsphere Stock Solution was immediately pipetted into well A1 of a Corning 96-well flat black plate.
  6. 100 μl of Microsphere Stock Solution was pipetted into A2 where it was mixed by pipetting up and down 3 times. Then 100μL of A2 was pipetted into A3 where it was mixed. This was continued down the plate until A11, where 100μL was taken out and binned. This whole process was repeated for B, C, and D.
  7. Each well was remixed by pipetting up and down before immediately being inserted into plate reader.
  8. The Abs600 of all wells was measured and data recorded.

A graph to show how the optical density changes with particle count. This data was obtained at a gain of 80 and temperature of 37 degrees Celsius. The R2 value for a linear fit to this data is 0.972. The graph shows the linear fit plotted on a log-log graph. Graph generated using MATLAB.



Calibration 3: Fluorescence Standard Curve (Fluorescein Protocol)

    Why? Fluorescence varies between instruments, so can’t be directly compared.

  1. The fluorescein kit tube (100μM) was spun down to make sure pellet was at the bottom of the tube.
  2. 1 mL of PBS was added to the fluorescein kit tube to create 10x fluorescein.
  3. The 10x fluorescein solution was diluted to 10μM by mixing 900μL of BPS with 10μL of the 10x fluorescein, this made 1x solution.
  4. Step 3 was repeated separately.
  5. One of the 1x solutions was diluted again to produce a 0.1x solution.
  6. 100μL of PBS was added to all wells except for A1, B1 … H1.
  7. 200μL of 1x fluorescein solution was added to wells A1, B1, C1, D1.
  8. 200μL of 0.1x fluorescein solution was added to wells E1, F1, G1, H1.
  9. Serial dilutions were performed across columns 1 to 11 by transferring 100μL between each well, leaving column 12 as a control.
  10. The fluorescence of all samples was measured in the plate reader at different gains and temperatures. Record data.
  11. The 1x and 0.1x results were compared to check for mistakes in the method.
  12. Data was imported into the InterLab spreadsheet provided.

A graph to show how the optical density changes with fluorescein concentration. This data was obtained at a gain of 80 and temperature of 37 degrees Celsius. The R2 value for a linear fit to this data is 0.9997. The graph shows the linear fit plotted on a log-log graph. Graph generated using MATLAB.



Cell Measurements

 For this part of the protocol, eight devices from the distribution kit were provided to transform into E. coli strain DH5α. These devices (BBa_R0040, BBa_I20270, BBa_J364000, BBa_J364001, BBa_J364002, BBa_J364007 BBa_J364008, and BBa_J364009) can be found in the iGEM directory. This process was split into a three day period; transforming, inoculation, and cell growth with assays. These cells were then grown to put through the FACS machine, in the final protocol for flow cytometry.

Flow Cytometry

 After the incubation of the two 96-well plates (0hrs and 6hrs), each sample was placed (146 in total) through a BD FACSAria III. Every well was pipetted into a plastic tube and read in the FACS machine. Each well had at least 10,000 events. The FCS files were then appropriately named - e.g. Cell samples: [Exeter]_[0]h_[A3]_[Device1].fcs - and kept together in a zip folder.

Colony Forming Units:
This was used to help produce a calibration between OD600 to CFU counts. This resulted in a cell concentration of the culture.
  1. In this experiment, only the two positive (BBa_I20270) and negative (BBa_R0040) control cultures were used. They were incubated the night before.
  2. A solution consisting of 100ml of LB with 71.4μl chloramphenicol was made up in a reliable measuring container.
  3. A 1:8 dilution of each overnight culture was made straight into the first four wells of the plate (25μl culture and 175μl LB).
  4. This was measured in the plate reader at 600nm and converted to OD by multiplying by the reference value obtained in Calibration 1 (2.0471).
  5. The amount of culture needed to make an optical density of 0.1 was calculated for each culture.
  6. These concentrations were then made up in four 2ml Eppendorf tubes to 1ml total volume before 200μl were placed in the wells and measured in the plate reader to make sure the OD was indeed 0.1.
  7. For each solution, 200μl were pipetted into three different wells so that three repeats were obtained for each of the four cultures.
  8. Serial dilutions were then carried out on each of the repeats as shown in the diagram opposite.
  9. The corresponding plates were spread and put in the incubator overnight at 37 degrees Celsius.
  10. The colony counts for plates which had fewer than 300 colonies were counted and recorded.
  11. This was then multiplied by the final dilution factor on each plate to obtain the CFU per ml of an OD600 culture.

A diagram showing the method for the serial dilutions in the Colony Forming Units section of InterLab. Taken from the iGEM 2018 InterLab protocol.


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