Section I: Provenance and Release

  1. Who did the actual work to acquire these measurements?
    The Interlab measurement study was conducted by Ariel Leyva-Hernandez, Yamini Naidu, and Stephanie Mao.
  2. What other people should be credited for these measurements? (i.e., who would be an author on any resulting publication. For example, your faculty advisor may have helped design the protocols that you ran.)
    The study was conducted in Dr. Farren Isaacs’s lab, with the mentoring of Natalie Ma.
  3. On what dates were the protocols run and the measurements taken? (this will often be a range of dates; make sure you say which data was taken at what times.)
    The construct protocols were finalized at the end of June, and the constructs were made over the span of July 21-September 3. The measurements were taken on September 9, 2014, and again on September 12, 2014.
  4. Do all persons involved consent to the inclusion of this data in publications derived from the iGEM interlab study?
    All persons mentioned give consent for their names to be included in the study.
  5. What is the model and manufacturer? How is it configured for your measurements? (e.g., light filters, illumination, amplification)
    A Synergy H1 Plate Reader (BioTek, Winooski, VT) was used for all optical measurements. The instrument uses a Xenon flash lamp as a light source. Fluorescence measurements were taken at single wavelengths ( λex = 485 ± 4 nm and λem = 528 ± 4 nm). A bottom fluorescence measurement was taken, which is configured with double grating monochromators. The reading speed for fluorescence and absorbance measurements for a 96-well plate is 11 seconds. Two photomultiplier tubes are used for detection: one for the monochromator system and another for the filter system.

Section II: Protocol
  1. What protocol did you use to prepare samples for measurement?
    Devices 2 and 3 were made using PCR with the specific promoter region in the forward and reverse overhangs, and BBa_E0240 as the template. The DNA was then DpnI digested and self-annealed by Gibson Assembly. This device was transformed into Mach1 competent cells, and colonies were screened using PCR before a plasmid miniprep was transformed into E.coli strain DH5α (F- endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG Φ80dlacZΔM15 Δ(lacZYA-argF)U169, hsdR17(rK- mK+), λ–).
  2. What sort of instrument did you use to acquire measurements?
    Measurements were conducted on the Synergy H1 Hybrid Multi-Mode Microplate Reader manufactured by Biotek. It was configured for monochromator fluorescence, monochromator absorbance, full-light luminescence, and time resolved fluorescence. The temperature control was set to 45°C. Measurements were taken with Gen5 data analysis software.
    Fluorescence Activity Assay Protocol
    1. Grow 1 mL culture with selection marker and inducer overnight.
    2. Transfer 150 µL culture into V-bottom plate.
    3. Spin down cells in plate with plate spinner set to a temperature 24-25ºC for 4 minutes. The plate shaker will pellet the cells in the v-bottom well over 4 minutes.
    4. Remove the supernatant and resuspend in 110 µL 1X PBS.
    5. Transfer 110 µL of 1X PBS into clear bottom plate.
    6. Transfer 10 µL of suspended cells into 100 µL fresh PBS in an adjacent well. Transfer 10 µL of second dilution into 90 µL of fresh PBS in an adjacent well.
    7. Using Synergy H1 plate reader (BioTek, Winooski, VT), measure optical density and fluorescence.
    8. Calculate expression of GFP
  3. What protocol did you use to take measurements?
    1. Shake plate for 10 seconds.
    2. easure optical density (OD) at 600 nm.
    3. Measure GFP fluorescence by exciting cells at 485 nm and detecting at 528 nm, with a bandpass of 4 nm on each side.
  4. What method is used to determine whether to include or exclude each sample from the data set?
    Fluorescence readings that diverged from the other samples of the same type by more than 50% were excluded from the data set; however, no such issues were encountered.
  5. What exactly were the controls that you used?
    During the growth phase of the cell cultures, the ancestral DH5α strain was also grown in LBmin and subject to the same antibiotic selection marker: kanamycin or chloramphenicol. Clear cultures after a day of incubation signified selection for the retention of the target plasmids. All cultures were seeded at the same time and allowed to grow for the same duration.
  6. What quantities were measured? (e.g., red fluorescence, green fluorescence, optical density)
    Green fluorescence and optical density were measured.
  7. How much time did it take to acquire each set of measurements?
    A time lapse of about five minutes occurred between each data set. The plates were seeded and incubated for 12 hours prior to measurement.
  8. How much does it cost to acquire a set of measurements?
    We are only considering the cost of the non-renewable materials, which were consumed during our measurements. Below is a chart of the resources used:
    Gibson Assembly mix$31.40
    LB and LB Plate$5.00
    96 well plate$2.00
  9. What are the practical limits on the number or rate of measurements taken with this instrument and protocol?
    The DH5α strain used was not a robust strain, and its growing time greatly impeded construct creation and measurement.

Section III: Measured Quantities
For each type of quantity measured (e.g., fluorescence, optical density), report on the following:
  1. Units:
    1. What are the units of the measurement?
      Fluorescence was measured in relative fluorescence units (RFU).
      Optical Density was measured in absorbance units (AU).
    2. What is the equivalent unit expressed as a combination of the seven SI base units?
      Absorbance in AU is a ratio, and thus has no SI units.
      RFU is an arbitrary fluorescence unit and cannot be compared.
  2. Precision:
    1. What is the range of possible measured values for this quantity, using your instrument as configured for these measurements? (e.g., a meter stick measures in the range of 0 to 1 meter)
      Dynamic range: 0-4 OD
    2. What are the significant figures for these measurement? (e.g., on a meter stick, it is common to measure to the nearest millimeter).
      0.0001 OD
    3. Is the precision the same across the entire range? If not, how does it differ?
    4. How did you determine these answers?
      Synergy H1 Specification Sheet
  3. Accuracy:
    1. When was the instrument last calibrated?
      March 2013
    2. How was the instrument calibrated?
      An accredited BioTek technician calibrated the machine against a known standard concentration gradient during initial setup of the machine.

Section IV: Measurements
  1. 1. For each sample, report:
    1. the identity of the sample
    2. each quantity directly measured
    3. each quantity derived from measurements (e.g., fluorescence/OD)

Table 1: Optical Density Measurements (AU)
DeviceDilution 1Dilution 2Dilution 3
Bba_J23101 + Bba_E02400.7270.1120.046
Bba_J23114 + Bba_E02400.7830.1390.047

Table 2: Fluorescence Measurements (RFU)
DeviceDilution 1Dilution 2Dilution 3
Bba_J23101 + Bba_E0240813788055166
Bba_J23114 + Bba_E02409353611521173

Table 3: Fluorescence Normalized by Optical Density (AFU/OD)
DeviceDilution 1Dilution 2Dilution 3
Bba_J23101 + Bba_E0240111937719203609
Bba_J23114 + Bba_E0240119458828853681

Graph 1: Fluorescence Normalized by Optical Density (AFU/OD)
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