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Experiment 3 - Cell measurement protocol Challenge

This year we plan to test protocols that will eventually be automated. For this reason, we will use 96-well plates instead of test tubes for culturing. Consequently, we want to evaluate how the performance of our plate culturing protocol compares to culturing in test tubes (e.g. 10 mL falcon tube) on a global scale. This version of the interlab protocol involves 2 hr. time interval measurements and incubation inside a microplate reader/incubator.

At the end of the experiment, you will have four plates to be measured. You will measure both fluorescence and absorbance in each plate.

Before performing the cell measurements, you need to perform all the calibration measurements. Please do not proceed unless you have completed the calibration protocol. 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 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. If you are absolutely unable to obtain the DH5-alpha strain, you may still participate in the InterLab study by contacting the Engineering Committee (engineering [at] igem [dot] org) to discuss your situation.

For all below indicated cell measurements, you must use the same type of plates and the same 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 type of plates, volumes, and settings, the measurements will not be valid.

Protocol summary: UPDATE You will transform the eight devices listed in Table 1 into E. coli K-12 DH5-alpha cells. The next day you will pick two colonies from each transformation (16 total) and use them to inoculate 5 mL overnight cultures (this step is still in tubes). Each of these 16 overnight cultures will be used to inoculate four wells in a 96-well plate (200uL each, 4 replicates) and one test tube (12 mL). You will measure how fluorescence and optical density develops over 6 hours by taking measurements at time point 0 hour and at time point 6 hours. Follow the protocol below and the visual instructions in Figure 1 and Figure 2.

Protocol Outputs:

Protocol Materials:

Table 1: Part Locations in Distribution Kit

Part Coordinate
BBa_J428100 Kit Plate 1 Well 12M
BBa_I20270 Kit Plate 1 Well 1A
BBa_J364000 Kit Plate 1 Well 1C
BBa_J364001 Kit Plate 1 Well 1E
BBa_J364002 Kit Plate 1 Well 1G
BBa_J364007 Kit Plate 1 Well 1I
BBa_J364008 Kit Plate 1 Well 1K
BBa_J364009 Kit Plate 1 Well 1M

Protocol Steps:

  1. Obtain 8 x Petri dish containing LB Agar + Chloramphenicol (34 ug/mL) growth medium for culturing transformant strains
  2. Transform Negative control DNA into E. coli DH5 alpha competent cells. Repeat for the remaining transformant DNA: Positive control (I20270), Test Device 1 (J364000), Test Device 2 (J364001), Test Device 3 (J364002), Test Device 4 (J364007), Test Device 5 (J364008), and Test Device 6 (J364009). Plate transformants on LB Agar + Chloramphenicol (34 ug/mL) transformant strains plates. Incubate overnight (for 16 hour) at 37.0°C.
  3. Obtain 16 x culture tubes to contain culture (day 1)
  4. Pick 2 colonies from each transformant strains plate.
  5. Inoculate 2 colonies of each transformant strains, for a total of 16 cultures. Inoculate each into 5.0mL of LB Broth + Chloramphenicol (34 ug/mL) in culture (day 1) and grow overnight (for 16.0 hour) at 37.0°C and 220 rpm.
  6. Obtain 16 x culture tubes to contain culture (day 2)
  7. Dilute each of 16 culture (day 1) samples with LB Broth + Chloramphenicol (34 ug/mL) into the culture tube at a 1:10 ratio and final volume of 5.0mL. Maintain at 4.0°C while performing dilutions. (This can be also performed on ice).
  8. Obtain 16 x 1.5 mL microfuge tubes to contain cultures (0 hr timepoint)
  9. Hold cultures (0 hr timepoint) on ice. This will prevent cell growth while transferring samples.
  10. Transfer 1.0mL of each of 16 culture (day 2) samples to wells <xarray.Dataset> Dimensions: () Data variables: empty of 1.5 mL microfuge tube containers to contain a total of 16 cultures (0 hr timepoint) samples. Maintain at 4.0°C during transfer. (This can be also performed on Ice).
  11. Measure baseline absorbance of culture (day 2) of cultures (0 hr timepoint) at 600.0nm.
  12. Obtain 16 x 50 ml conical tubes to contain back-diluted culture The conical tube should be opaque, amber-colored, or covered with foil.
  13. Back-dilute each of 16 culture (day 2) samples to a target OD of 0.02 using LB Broth + Chloramphenicol (34 ug/mL) as diluent to a final volume of 40.0mL. Maintain at 4.0°C while performing dilutions.

Fig 1: Visual representation of protocol

  1. Obtain 16 x 50 ml conical tubes to contain Tubes 1, 2 and 3 The conical tubes should be opaque, amber-colored, or covered with foil.
  2. Obtain 16 x 50 ml conical tubes to contain Tube 2 The conical tubes should be opaque, amber-colored, or covered with foil.
  3. Obtain 16 x 50 ml conical tubes to contain Tube 3 The conical tubes should be opaque, amber-colored, or covered with foil.
  4. Transfer 12.0mL of each of 16 back-diluted culture samples to wells <xarray.Dataset> Dimensions: () Data variables: empty of 50 ml conical tube containers to contain a total of 16 Tubes 1, 2 and 3 samples. Maintain at 4.0°C during transfer.
  5. Transfer 12.0mL of each of 16 back-diluted culture samples to wells <xarray.Dataset> Dimensions: () Data variables: empty of 50 ml conical tube containers to contain a total of 16 Tube 2 samples. Maintain at 4.0°C during transfer.
  6. Transfer 12.0mL of each of 16 back-diluted culture samples to wells <xarray.Dataset> Dimensions: () Data variables: empty of 50 ml conical tube containers to contain a total of 16 Tube 3 samples. Maintain at 4.0°C during transfer.
  7. Provision a container named plate 1 such as: NEST96WellPlate, Corning96WellPlate360uLFlat.
  8. Hold all None samples on ice.
  9. Transfer 200.0uL of each back-diluted culture sample to 96 well plate plate 1 in the wells indicated in the plate layout. Maintain at 4.0°C during transfer.
  10. Transfer 200.0uL of LB Broth + Chloramphenicol (34 ug/mL) sample to wells {'A1': None, 'B1': None, 'C1': None, 'D1': None, 'E1': None, 'F1': None, 'G1': None, 'H1': None, 'A2': None, 'B2': None, 'C2': None, 'D2': None, 'E2': None, 'F2': None, 'G2': None, 'H2': None, 'A3': None, 'B3': None, 'C3': None, 'D3': None, 'E3': None, 'F3': None, 'G3': None, 'H3': None, 'A4': None, 'B4': None, 'C4': None, 'D4': None, 'E4': None, 'F4': None, 'G4': None, 'H4': None, 'A5': None, 'B5': None, 'C5': None, 'D5': None, 'E5': None, 'F5': None, 'G5': None, 'H5': None, 'A6': None, 'B6': None, 'C6': None, 'D6': None, 'E6': None, 'F6': None, 'G6': None, 'H6': None, 'A7': None, 'B7': None, 'C7': None, 'D7': None, 'E7': None, 'F7': None, 'G7': None, 'H7': None, 'A8': None, 'B8': None, 'C8': None, 'D8': None, 'E8': None, 'F8': None, 'G8': None, 'H8': None, 'A9': None, 'B9': None, 'C9': None, 'D9': None, 'E9': None, 'F9': None, 'G9': None, 'H9': None, 'A10': None, 'B10': None, 'C10': None, 'D10': None, 'E10': None, 'F10': None, 'G10': None, 'H10': None, 'A11': None, 'B11': None, 'C11': None, 'D11': None, 'E11': None, 'F11': None, 'G11': None, 'H11': None, 'A12': None, 'B12': None, 'C12': None, 'D12': None, 'E12': None, 'F12': None, 'G12': None, 'H12': None, 'A2:D2': 'http://igem.org/engineering/transformant1', 'E2:H2': 'http://igem.org/engineering/transformant2', 'A3:D3': 'http://igem.org/engineering/transformant3', 'E3:H3': 'http://igem.org/engineering/transformant4', 'A4:D4': 'http://igem.org/engineering/transformant5', 'E4:H4': 'http://igem.org/engineering/transformant6', 'A5:D5': 'http://igem.org/engineering/transformant7', 'E5:H5': 'http://igem.org/engineering/transformant8', 'A7:D7': 'http://igem.org/engineering/transformant1', 'E7:H7': 'http://igem.org/engineering/transformant2', 'A8:D8': 'http://igem.org/engineering/transformant3', 'E8:H8': 'http://igem.org/engineering/transformant4', 'A9:D9': 'http://igem.org/engineering/transformant5', 'E9:H9': 'http://igem.org/engineering/transformant6', 'A10:D10': 'http://igem.org/engineering/transformant7', 'E10:H10': 'http://igem.org/engineering/transformant8'} of 96 well plate plate 1. Maintain at 4.0°C during transfer. These samples are blanks.

Fig 2: Plate layout

  1. Measure 0 hr absorbance timepoint of plate 1 at 600.0nm.
  2. Measure 0 hr fluorescence timepoint of plate 1 with excitation wavelength of 488.0nm and emission filter of 530.0nm and 30.0nm bandpass.
  3. Cover plate 1 samples in 96 well plate with your choice of material to prevent evaporation.
  4. Incubate all plate 1 samples for 6.0 hour at 37.0°C at 220 rpm.
  5. Measure absorbance timepoint of plate 1 at 600.0nm at timepoints 2.0 hour, 4.0 hour, 6.0 hour.
  6. Measure fluorescence timepoint of plate 1 with excitation wavelength of 488.0nm and emission filter of 530.0nm and 30.0nm bandpass at timepoints 2.0 hour, 4.0 hour, 6.0 hour.
  7. Incubate Tubes 1, 2 and 3 for 2.0 hour at 37.0°C at 220 rpm.
  8. Hold Tubes 1, 2 and 3 on ice. Reserve until the end of the experiment for absorbance and fluorescence measurements.
  9. Incubate Tube 2 for 4.0 hour at 37.0°C at 220 rpm.
  10. Hold Tube 2 on ice. Reserve until the end of the experiment for absorbance and fluorescence measurements.
  11. Incubate Tube 3 for 6.0 hour at 37.0°C at 220 rpm.
  12. Hold Tube 3 on ice. Reserve until the end of the experiment for absorbance and fluorescence measurements.
  13. Provision a container named Plates 2, 3, and 4 such as: NEST96WellPlate, Corning96WellPlate360uLFlat.
  14. Transfer 200.0uL of each Tubes 1, 2 and 3 sample to 96 well plate Plates 2, 3, and 4 in the wells indicated in the plate layout. Maintain at 4.0°C during transfer.
  15. Transfer 200.0uL of LB Broth + Chloramphenicol (34 ug/mL) sample to wells {'A1': None, 'B1': None, 'C1': None, 'D1': None, 'E1': None, 'F1': None, 'G1': None, 'H1': None, 'A2': None, 'B2': None, 'C2': None, 'D2': None, 'E2': None, 'F2': None, 'G2': None, 'H2': None, 'A3': None, 'B3': None, 'C3': None, 'D3': None, 'E3': None, 'F3': None, 'G3': None, 'H3': None, 'A4': None, 'B4': None, 'C4': None, 'D4': None, 'E4': None, 'F4': None, 'G4': None, 'H4': None, 'A5': None, 'B5': None, 'C5': None, 'D5': None, 'E5': None, 'F5': None, 'G5': None, 'H5': None, 'A6': None, 'B6': None, 'C6': None, 'D6': None, 'E6': None, 'F6': None, 'G6': None, 'H6': None, 'A7': None, 'B7': None, 'C7': None, 'D7': None, 'E7': None, 'F7': None, 'G7': None, 'H7': None, 'A8': None, 'B8': None, 'C8': None, 'D8': None, 'E8': None, 'F8': None, 'G8': None, 'H8': None, 'A9': None, 'B9': None, 'C9': None, 'D9': None, 'E9': None, 'F9': None, 'G9': None, 'H9': None, 'A10': None, 'B10': None, 'C10': None, 'D10': None, 'E10': None, 'F10': None, 'G10': None, 'H10': None, 'A11': None, 'B11': None, 'C11': None, 'D11': None, 'E11': None, 'F11': None, 'G11': None, 'H11': None, 'A12': None, 'B12': None, 'C12': None, 'D12': None, 'E12': None, 'F12': None, 'G12': None, 'H12': None} of 96 well plate Plates 2, 3, and 4. Maintain at 4.0°C during transfer. These samples are blanks.
  16. Measure absorbance timepoint of Plates 2, 3, and 4 at 600.0nm.
  17. Measure fluorescence timepoint of Plates 2, 3, and 4 with excitation wavelength of 488.0nm and emission filter of 530.0nm and 30.0nm bandpass.
  18. Import data into the provided Excel file: baseline absorbance of culture (day 2) measurements of cultures (0 hr timepoint), 0 hr absorbance timepoint measurements of plate 1, 0 hr fluorescence timepoint measurements of plate 1, absorbance timepoint measurements of plate 1 at timepoints 2.0 hour, 4.0 hour, 6.0 hour, fluorescence timepoint measurements of plate 1 at timepoints 2.0 hour, 4.0 hour, 6.0 hour, absorbance timepoint measurements of Plates 2, 3, and 4, fluorescence timepoint measurements of Plates 2, 3, and 4.

Timestamp: 2023-03-11 16:06:01.091432 Protocol version: 1.2b