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Cell measurement protocol

Description:

Prior to performing the cell measurements you should perform all three of the calibration measurements. Please do not proceed unless you have completed the three calibration protocols. Completion of the calibrations will ensure that you understand the measurement process and that you can take the cell measurements under the same conditions. For the sake of consistency and reproducibility, we are requiring all teams to use E. coli K-12 DH5-alpha. If you do not have access to this strain, you can request streaks of the transformed devices from another team near you, and this can count as a collaboration as long as it is appropriately documented on both teams' wikis. If you are absolutely unable to obtain the DH5-alpha strain, you may still participate in the InterLab study by contacting the Measurement Committee (measurement at igem dot org) to discuss your situation.

For all of these cell measurements, you must use the same plates and volumes that you used in your calibration protocol. You must also use the same settings (e.g., filters or excitation and emission wavelengths) that you used in your calibration measurements. If you do not use the same plates, volumes, and settings, the measurements will not be valid.

Protocol Materials:

Protocol Inputs:

Protocol Outputs:

  • baseline absorbance of culture (day 2) measurements of culture (day 2)
  • 0 hr absorbance timepoint measurements of plate 1
  • 0 hr fluorescence timepoint measurements of plate 1
  • 6 hr absorbance timepoint measurements of plate 1
  • 6 hr fluorescence timepoint measurements of plate 1
  • 6 hr absorbance timepoint measurements of plate 2
  • 6 hr fluorescence timepoint measurements of plate 2

Steps

  1. Transform Negative control DNA into E. coli DH5 alpha and plate transformants on LB Broth+chloramphenicol. Repeat for the remaining transformant DNA: Positive control, Test Device 1, Test Device 2, Test Device 3, Test Device 4, Test Device 5, and Test Device 6.
  2. Provision 16 x culture tubes to contain culture (day 1)
  3. Inoculate E. coli DH5 alpha+Negative control transformant into 5.0 milliliter of LB Broth+chloramphenicol in culture (day 1) and grow for 16.0 hour at 37.0 degree Celsius and 220.0 rpm. Repeat this procedure for the other inocula: E. coli DH5 alpha+Positive control transformant, E. coli DH5 alpha+Test Device 1 transformant, E. coli DH5 alpha+Test Device 2 transformant, E. coli DH5 alpha+Test Device 3 transformant, E. coli DH5 alpha+Test Device 4 transformant, E. coli DH5 alpha+Test Device 5 transformant, and E. coli DH5 alpha+Test Device 6 transformant. Inoculate 2 replicates for each transformant, for a total of 16 cultures.
  4. Provision 16 x culture tubes to contain culture (day 2)
  5. Dilute each of 16 culture (day 1) samples with LB Broth+chloramphenicol into the culture tube at a 1:10 ratio and final volume of 5.0 milliliter. Maintain at 4.0 degree Celsius while performing dilutions.
  6. Measure baseline absorbance of culture (day 2) of culture (day 2) at 600.0 nanometer.
  7. Provision 16 x 50 ml conical tubes to contain back-diluted culture The conical tube should be opaque, amber-colored, or covered with foil.
  8. Back-dilute each of 16 culture (day 2) samples to a target OD of 0.02 using LB Broth+chloramphenicol as diluent to a final volume of 12.0 milliliter. Maintain at 4.0 degree Celsius while performing dilutions.

  1. Provision 16 x 1.5 mL microfuge tubes to contain cultures (0 hr timepoint)
  2. Provision a 96 well microplate to contain plate 1
  3. Hold cultures (0 hr timepoint) at 4.0 degree Celsius. This will prevent cell growth while transferring samples.
  4. Hold plate 1 at 4.0 degree Celsius.
  5. Transfer 1.0 milliliter of each of 16 back-diluted culture samples to 1.5 mL microfuge tube containers to contain a total of 16 cultures (0 hr timepoint) samples. Maintain at 4.0 degree Celsius during transfer.
  6. Transfer 100.0 microliter of each cultures (0 hr timepoint) sample to 96 well microplate plate 1 in the wells indicated in the plate layout. Maintain at 4.0 degree Celsius during transfer.
  7. Transfer 100.0 microliter of LB Broth+chloramphenicol sample to wells A1:H1, A10:H10, A12:H12 of 96 well microplate plate 1. Maintain at 4.0 degree Celsius during transfer. These samples are blanks.

  1. Measure 0 hr absorbance timepoint of plate 1 at 600.0 nanometer.
  2. Measure 0 hr fluorescence timepoint of plate 1 with excitation wavelength of 488.0 nanometer and emission filter of 530.0 nanometer and 30.0 nanometer bandpass
  3. Cover plate 1 samples in 96 well microplate with your choice of material to prevent evaporation.
  4. Incubate all back-diluted culture samples for 6.0 hour at 37.0 degree Celsius at 220.0.
  5. Incubate all plate 1 samples for 6.0 hour at 37.0 degree Celsius at 220.0.
  6. Hold all cultures (0 hr timepoint) samples at 4.0 degree Celsius. This will inhibit cell growth during the subsequent pipetting steps.
  7. Hold all plate 1 samples at 4.0 degree Celsius. This will inhibit cell growth during the subsequent pipetting steps.
  8. Provision 16 x 1.5 mL microfuge tubes to contain 6hr timepoint
  9. Provision a 96 well microplate to contain plate 2
  10. Hold 6hr timepoint at 4.0 degree Celsius. This will prevent cell growth while transferring samples.
  11. Hold plate 2 at 4.0 degree Celsius.
  12. Transfer 1.0 milliliter of each of 16 back-diluted culture samples to 1.5 mL microfuge tube containers to contain a total of 16 6hr timepoint samples. Maintain at 4.0 degree Celsius during transfer.
  13. Transfer 100.0 microliter of each 6hr timepoint sample to 96 well microplate plate 2 in the wells indicated in the plate layout. Maintain at 4.0 degree Celsius during transfer.
  14. Transfer 100.0 microliter of LB Broth+chloramphenicol sample to wells A1:H1, A10:H10, A12:H12 of 96 well microplate plate 2. Maintain at 4.0 degree Celsius during transfer. These are the blanks.
  15. Perform a brief centrifugation on 96 well microplate containing plate 1 samples. This will prevent cross-contamination when removing the seal.
  16. Remove the seal from 96 well microplate containing plate 1 samples.
  17. Measure 6 hr absorbance timepoint of plate 1 at 600.0 nanometer.
  18. Measure 6 hr fluorescence timepoint of plate 1 with excitation wavelength of 485.0 nanometer and emission filter of 530.0 nanometer and 30.0 nanometer bandpass
  19. Measure 6 hr absorbance timepoint of plate 2 at 600.0 nanometer.
  20. Measure 6 hr fluorescence timepoint of plate 2 with excitation wavelength of 485.0 nanometer and emission filter of 530.0 nanometer and 30.0 nanometer bandpass
  21. Import data for baseline absorbance of culture (day 2) measurements of culture (day 2), 0 hr absorbance timepoint measurements of plate 1, 0 hr fluorescence timepoint measurements of plate 1, 6 hr absorbance timepoint measurements of plate 1, 6 hr fluorescence timepoint measurements of plate 1, 6 hr absorbance timepoint measurements of plate 2, 6 hr fluorescence timepoint measurements of plate 2 into provided Excel file.

Timestamp: 2022-04-24 22:42:28.454856--- Protocol version: 1.0b