Team:BostonU/InterLab
This page documents BostonU 2018's contribution to the Fifth International InterLaboratory Measurement Study in synthetic biology, or "InterLab 5." Currently, it is difficult to conduct reliable and repeatable measurements across different instruments in different labs. This acts as a barrier to collaboration and progress in the synthetic biology community. InterLab is a crowdsourced research study aiming to standardize measurement tools across the synthetic biology community by developing new measurement procedures for green fluorescent protein (GFP), a common marker in synthetic biology.
Compounding the difficulty of repeating measurements across labs is the lack of absolute units of fluorescence. The vast majority of published measurements are arbitrary fluorescence units (AFU or AU), which are defined entirely by procedure. 10 AFU from one lab cannot be directly compared to 1000 AFU from another, without analyzing the exact procedures of each experiment. The InterLab protocol aims to address these issues by providing researchers with a detailed protocol and data analysis form that yields absolute units for measurements of GFP in a plate reader. The goal of the fifth InterLab in particular is to reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell counts or colony-forming units instead of optical density.
Below is a summary of the work BostonU did to contribute to the InterLab study, including protocols, raw and analyzed data, and specifications of the instruments used in the Khalil Lab at Boston University. The original protocols defined by the InterLab study can be found here.
Calibration
The table below lists the relevant qualities of our plate reader:
| Instrument Property | Property Value |
|---|---|
| Instrument Type | plate reader |
| Brand and Model | SpectraMax M5 |
| Absorbance and Fluorescence? | both |
| Top or bottom optics? | bottom optics |
| Pathlength correction? | yes; can be disabled |
| GFP bandpass filter | 530 nm / 30 nm, 30 nm width |
| GFP excitation wavelength | 485 nm |
| GFP emission wavelength | 525 nm |
| Temperature Setting | room temperature (all measurements) |
The table below lists the relevant qualities of our 96-well plates:
| Plate Property | Property Value |
|---|---|
| Plate Color | clear |
| Plate Bottom | flat |
Optical Density (OD600) Reference Point - LUDOX
- Add 100 µL LUDOX 100% into wells A1, B1, C1, D1.
- Add 100 µL of dH20 into A2, B2, C2, D2.
- Measure absorbance 600 nm (AB600) of all samples.
Below are the AB600 measurements of the prepared plate, and brief analysis of those measurements:
| Item | LUDOX CL-X | H20 |
|---|---|---|
| Replicate 1 | 0.107 | 0.090 |
| Replicate 2 | 0.108 | 0.089 |
| Replicate 3 | 0.111 | 0.089 |
| Replicate 4 | 0.106 | 0.089 |
| Arithmetic Mean | 0.108 | 0.089 |
| Corrected Abs600 | 0.019 | |
| Reference OD600 | 0.063 | |
| OD600/Abs600 | 3.360 |
Particle Standard Curve - Microsphere
The silica microspheres used in this calibration step are similar in size and optical characteristics to E. coli cells. This calibration step will construct a standard curve of particle concentration which can be usedto convert Abs600 measurements to estimated numbers of cells.
All measurements in this step were taken without pathlength correction and at room temperature.
First, obtain a microsphere stock solution:
- Obtain the tube labeled "Silica Beads" from the measurement kit and vortex vigorously for 30 seconds.
- Immediately pipet 96 µL of microspheres into a 1.5 mL eppendorf tube.
- Add 904 µL of ddH20 to the microspheres
- Vortex well.
Whenever working with microspheres, it is desirable to continuously "fluff" the mixture by forcefully pippeting up and down. This resuspends the microspheres, which tend to sink rapidly to the bottom of any mixture.
Conduct a 12-step serial dilution of microspheres in a 96-well plate:
- Add 100 µL of ddH20 to wells A2-12, B2-12, C2-12 and D2-12.
- Resuspend your microsphere stock solution by vortexing or pippeting vigorously. Add 200 µL of microsphere stock to well A1
- Transfer 100 µL of well A1 into well A2.
- Continue transfering 100 µL of the previous well into the next one, until you pipette 100 µL from well A10 into well A11.
- Pipette 100 µL from well A11 to liquid waste.
- Repeat steps 2 through 5 for row B, C and D.
- Fluff (pipette up and down) every well in the plate, then read the Abs600 of the well samples.
Our Abs600 data and analysis from following the above protocol is below:
| Number of Particles | 2.35E+08 | 1.18E+08 | 5.88E+07 | 2.94E+07 | 1.47E+07 | 7.35E+06 | 3.68E+06 | 1.84E+06 | 9.19E+05 | 4.60E+05 | 2.30E+05 | 0 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Replicate 1 | 0.970 | 0.566 | 0.345 | 0.224 | 0.176 | 0.133 | 0.112 | 0.108 | 0.098 | 0.094 | 0.093 | 0.089 |
| Replicate 2 | 1.304 | 0.613 | 0.389 | 0.215 | 0.152 | 0.125 | 0.111 | 0.103 | 0.097 | 0.092 | 0.091 | 0.089 |
| Replicate 3 | 1.338 | 0.895 | 0.607 | 0.440 | 0.197 | 0.171 | 0.146 | 0.115 | 0.104 | 0.099 | 0.094 | 0.089 |
| Replicate 4 | 1.504 | 1.051 | 0.606 | 0.364 | 0.239 | 0.153 | 0.133 | 0.105 | 0.099 | 0.096 | 0.094 | 0.089 |
| Aritd. Mean | 1.279 | 0.781 | 0.487 | 0.311 | 0.191 | 0.146 | 0.126 | 0.108 | 0.100 | 0.095 | 0.093 | 0.089 |
| Aritd. Std.Dev. | 0.224 | 0.231 | 0.139 | 0.110 | 0.037 | 0.021 | 0.017 | 0.005 | 0.003 | 0.003 | 0.001 | 0.000 |
| Aritd. Net Mean | 1.190 | 0.692 | 0.398 | 0.222 | 0.102 | 0.057 | 0.037 | 0.019 | 0.011 | 0.006 | 0.004 | |
| Mean particles/Abs600 | 1.98E+08 | 1.70E+08 | 1.48E+08 | 1.33E+08 | 1.44E+08 | 1.30E+08 | 1.01E+08 | 9.80E+07 | 8.75E+07 | 7.35E+07 | 5.74E+07 | |
| Mean of med-high levels: | 1.45E+08 |
Fluoresence standard curve - FIuorescein
All plate reader settings for measurements in this section were as labeled above, in the Overview section. First, prepare the fluorescein stock solution as below:
- Resuspend 10X fluorescein pellet in 1 mL of 1X phosphate buffer solution (PBS).
- Add 100 µL of 10X fluorescein stock to 900 µL of PBS to make a 1X fluorescein master mix.
Now, prepare the serial dilution of fluorescein in a 96-well plate as below:
- Add 100 µL of phosphate buffer solution (PBS) to wells A2-12, B2-12, C2-12 and D2-12.
- Add 200 µL of 1X fluorescein stock to well A1
- Transfer 100 µL of well A1 into well A2.
- Continue transfering 100 µL of the previous well into the next one, until you pipette 100 µL from well A10 into well A11.
- Pipette 100 µL from well A11 to liquid waste.
- Repeat steps 2 through 5 for row B, C and D.
- Measure the fluorescence of the well samples.
The plate readings for the fluorescein serial dilution described above can be found in the table below, along with a brief analysis.
| Fluorescein uM | 10.00 | 5 | 2.5 | 1.25 | 0.625 | 0.313 | 0.156 | 0.078 | 0.039 | 0.0195 | 0.0098 | 0 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Replicate 1 | 5.500E+04 | 3.300E+04 | 1.800E+04 | 9.487E+03 | 4.037E+03 | 2.000E+03 | 1.128E+03 | 5.743E+02 | 2.909E+02 | 1.546E+02 | 8.747E+01 | 1.542E+01 |
| Replicate 2 | 5.600E+04 | 3.300E+04 | 1.800E+04 | 9.126E+03 | 4.612E+03 | 2.455E+03 | 1.576E+03 | 6.533E+02 | 3.418E+02 | 1.744E+02 | 9.629E+01 | 1.481E+01 |
| Replicate 3 | 5.400E+04 | 3.200E+04 | 1.800E+04 | 8.857E+03 | 4.166E+03 | 2.086E+03 | 1.070E+03 | 5.283E+02 | 2.737E+02 | 1.420E+02 | 7.930E+01 | 1.504E+01 |
| Replicate 4 | 5.300E+04 | 3.100E+04 | 1.700E+04 | 8.819E+03 | 4.043E+03 | 2.050E+03 | 1.018E+03 | 5.121E+02 | 2.673E+02 | 1.397E+02 | 7.864E+01 | 1.550E+01 |
| Arith. Mean | 5.450E+04 | 3.225E+04 | 1.775E+04 | 9.072E+03 | 4.214E+03 | 2.148E+03 | 1.198E+03 | 5.670E+02 | 2.934E+02 | 1.527E+02 | 8.542E+01 | 1.519E+01 |
| Arith. Std.Dev. | 1.291E+03 | 9.574E+02 | 5.000E+02 | 3.083E+02 | 2.715E+02 | 2.078E+02 | 2.560E+02 | 6.330E+01 | 3.374E+01 | 1.591E+01 | 8.282E+00 | 3.241E-01 |
| Arith. Net Mean | 5.448E+04 | 3.223E+04 | 1.773E+04 | 9.057E+03 | 4.199E+03 | 2.133E+03 | 1.183E+03 | 5.518E+02 | 2.782E+02 | 1.375E+02 | 7.023E+01 | |
| uM Fluorescein/a.u. | 1.84E-04 | 1.55E-04 | 1.41E-04 | 1.38E-04 | 1.49E-04 | 1.47E-04 | 1.32E-04 | 1.42E-04 | 1.40E-04 | 1.42E-04 | 1.39E-04 | |
| Mean uM fluorescein / a.u.: | 1.46E-04 | |||||||||||
| MEFL / a.u.: | 8.79E+08 |
Cell Measurements
The following section details the work BostonU did to construct and test the eight test devices outlined in the InterLab protocol. It is important to note here that BostonU used NEB 5-alpha High Efficiency competent cells instead of Thermo Fischer's 5-alpha competent cells as parent strains for these test devices. The test devices, and their primary transformed-in plasmid construct, can be found in the table below:
| Device | Part Number | Plate | Location |
|---|---|---|---|
| Positive control | BBa_I20270 | Kit Plate 7 | Well 2B |
| Negative control | BBa_R0040 | Kit Plate 7 | Well 2D |
| Test Device 1 | BBa_J364000 | Kit Plate 7 | Well 2F |
| Test Device 2 | BBa_J364001 | Kit Plate 7 | Well 2H |
| Test Device 3 | BBa_J364002 | Kit Plate 7 | Well 2J |
| Test Device 4 | BBa_J364007 | Kit Plate 7 | Well 2L |
| Test Device 5 | BBa_J364008 | Kit Plate 7 | Well 2N |
| Test Device 6 | BBa_J364009 | Kit Plate 7 | Well 2P |
Protocols
For the purposes of constructing these test devices, the iGEM standard transformation protocol was used with slight variations. As stated above, we used NEB 5-alpha High Efficiency competent cells. Additionally, the rescue step (i.e. step 11) was reduced from 1 hour to 30 minutes, and the competent cell efficiency was not verified in step 16.
After these transformed cells grow overnight, two colonies from each plate were picked into 2 mL of LB+Chloramphenicol in a 24-well block. They were left to grow up over 8 hours. After these 8 hours, 500 µL of cells were removed from each well and used to make glycerol stock. This stock was used to grow more of each sample over night, for use in the next day's measurement protocol.
The next day, the Abs600 of each culture was measured, and each culture was diluted to 0.02 Abs600 in 4 mL of LB+Chloramphenicol in individual 12 mL aerated cell culture tubes. These tubes were covered with aluminum foil. This differs from the protocol outlined in the InterLab protocol packet, which calls for the same dilution in 12 mL of media in 50 mL falcon tubes. This change was made for three reasons: 50 mL falcon tubes cannot be used in our 30-degree shaking incubator, we did not have enough LB+Chloramphenicol liquid media for 8 tubes of 12 mL cultures, and cell growth should be faster in an oxygenated culture tube.
400 µL of each culture sample was removed at time point 0 and placed into a 96-well plate. The fluorescence and Abs600 of these 16 samples was immediately read in our plate reader and recorded. After 6 hours of growth, 400 µL of each sample were pipetted into the same 96-well plate, and the fluorescence and Abs600 of these samples was read and recorded. See the layout of this plate in the image below:
Data
This section contains the raw and analyzed data from the experiment above.
Raw Data
Fluorescence Raw Readings, Hour 0
| Hour 0: | Neg. Control | Pos. Control | Device 1 | Device 2 | Device 3 | Device 4 | Device 5 | Device 6 | LB + Chlor (blank) |
|---|---|---|---|---|---|---|---|---|---|
| Colony 1, Replicate 1 | 221.39 | 371.44 | 1325.7 | 509.48 | 173.63 | 1106.7 | 1356 | 408.96 | 173.82 |
| Colony 1, Replicate 2 | 211.41 | 371.99 | 1135.5 | 465.15 | 190.32 | 1216.1 | 1699.3 | 415.1 | 166.14 |
| Colony 1, Replicate 3 | 204.75 | 364.74 | 1120 | 536.93 | 198.28 | 1276.1 | 1841.6 | 413.91 | 165.88 |
| Colony 1, Replicate 4 | 206.9 | 357.66 | 1035.2 | 49.04 | 168.75 | 1077.9 | 1781.3 | 361.98 | 140.56 |
| Colony 2, Replicate 1 | 209 | 288.45 | 1865.1 | 520.35 | 165.67 | 1198.3 | 1653.5 | 375.02 | 154.55 |
| Colony 2, Replicate 2 | 218.89 | 293.61 | 1800.1 | 592.37 | 261.24 | 1224.2 | 1793.1 | 354.68 | 126.36 |
| Colony 2, Replicate 3 | 213.23 | 290.46 | 1293.6 | 607.6 | 198.65 | 1423.6 | 1412.8 | 359.21 | 148.38 |
| Colony 2, Replicate 4 | 223.81 | 299.04 | 2073.2 | 634.02 | 204.8 | 1635.5 | 1785.4 | 418.37 | 151.71 |
Fluorescence Raw Readings, Hour 6
| Hour 6: | Neg. Control | Pos. Control | Device 1 | Device 2 | Device 3 | Device 4 | Device 5 | Device 6 | LB + Chlor (blank) |
|---|---|---|---|---|---|---|---|---|---|
| Colony 1, Replicate 1 | 199.84 | 3025.7 | 4727.8 | 5875.6 | 418.78 | 9182.3 | 2795.2 | 2425.6 | 18.9 |
| Colony 1, Replicate 2 | 185.9 | 2920.8 | 3848.2 | 5008.9 | 369.36 | 7933.8 | 2608 | 1922.6 | 195.08 |
| Colony 1, Replicate 3 | 190.38 | 2813.3 | 3759.9 | 4829.4 | 331.3 | 7154.9 | 2514.5 | 1977.2 | 192.71 |
| Colony 1, Replicate 4 | 91.51 | 2755.6 | 3401.6 | 3994.7 | 321.48 | 6315.8 | 2232.9 | 1947.2 | 189.6 |
| Colony 2, Replicate 1 | 188.83 | 2555.6 | 4867.1 | 5074.1 | 344.61 | 10000 | 2176.5 | 1997.5 | 184.31 |
| Colony 2, Replicate 2 | 191.8 | 2568.3 | 4811.5 | 4910.4 | 326.04 | 11000 | 1916.9 | 2097.7 | 163.58 |
| Colony 2, Replicate 3 | 195.66 | 2206 | 4657.6 | 5261.3 | 337.04 | 11000 | 2005.5 | 2213 | 216.81 |
| Colony 2, Replicate 4 | 197 | 2927.2 | 6051.8 | 5803.2 | 357.57 | 11000 | 2465.7 | 2033.5 | 177.17 |
Abs600 Raw Readings, Hour 0
| Hour 0: | Neg. Control | Pos. Control | Device 1 | Device 2 | Device 3 | Device 4 | Device 5 | Device 6 | LB + Chlor (blank) |
|---|---|---|---|---|---|---|---|---|---|
| Colony 1, Replicate 1 | 0.281 | 0.302 | 0.314 | 0.299 | 0.288 | 0.298 | 0.313 | 0.288 | 0.127 |
| Colony 1, Replicate 2 | 0.282 | 0.301 | 0.319 | 0.289 | 0.289 | 0.31 | 0.32 | 0.285 | 0.33 |
| Colony 1, Replicate 3 | 0.305 | 0.302 | 0.329 | 0.305 | 0.282 | 0.306 | 0.302 | 0.283 | 0.131 |
| Colony 1, Replicate 4 | 0.299 | 0.312 | 0.305 | 0.293 | 0.269 | 0.294 | 0.296 | 0.287 | 0.118 |
| Colony 2, Replicate 1 | 0.279 | 0.273 | 0.419 | 0.321 | 0.293 | 0.345 | 0.29 | 0.303 | 0.123 |
| Colony 2, Replicate 2 | 0.273 | 0.273 | 0.414 | 0.312 | 0.274 | 0.322 | 0.283 | 0.286 | 0.12 |
| Colony 2, Replicate 3 | 0.271 | 0.27 | 0.289 | 0.321 | 0.271 | 0.322 | 0.311 | 0.286 | 0.119 |
| Colony 2, Replicate 4 | 0.275 | 0.28 | 0.396 | 0.324 | 0.283 | 0.33 | 0.282 | 0.29 | 0.116 |
Abs600 Raw Readings, Hour 6
| Hour 6: | Neg. Control | Pos. Control | Device 1 | Device 2 | Device 3 | Device 4 | Device 5 | Device 6 | LB + Chlor (blank) |
|---|---|---|---|---|---|---|---|---|---|
| Colony 1, Replicate 1 | 1.583 | 1.634 | 0.998 | 1.595 | 1.606 | 1.533 | 0.618 | 1.583 | 0.099 |
| Colony 1, Replicate 2 | 1.599 | 1.622 | 1.023 | 1.618 | 1.61 | 1.53 | 0.71 | 1.587 | 0.106 |
| Colony 1, Replicate 3 | 1.609 | 1.636 | 1.043 | 1.626 | 1.625 | 1.538 | 0.699 | 1.576 | 0.105 |
| Colony 1, Replicate 4 | 1.606 | 1.629 | 1.018 | 1.625 | 1.624 | 1.572 | 0.758 | 1.578 | 0.101 |
| Colony 2, Replicate 1 | 1.578 | 1.632 | 1.115 | 1.626 | 1.593 | 1.561 | 0.637 | 1.601 | 0.109 |
| Colony 2, Replicate 2 | 1.58 | 1.637 | 1.065 | 1.648 | 1.601 | 1.576 | 0.658 | 1.643 | 0.111 |
| Colony 2, Replicate 3 | 1.596 | 1.609 | 1.102 | 1.659 | 1.614 | 1.561 | 0.621 | 1.623 | 0.112 |
| Colony 2, Replicate 4 | 1.569 | 1.643 | 1.076 | 1.623 | 1.628 | 1.592 | 0.642 | 1.656 | 0.1 |
Analyzed Data
µThis data is all imported from the InterLab_2018_BostonU spreadsheet, where the formulas are found.
µM Fluorescein / OD, Hour 0
| Hour 0: | Neg. Control | Pos. Control | Device 1 | Device 2 | Device 3 | Device 4 | Device 5 | Device 6 |
| Colony 1, Replicate 1 | 0.013 | 0.049 | 0.267 | 0.085 | 0.000 | 0.237 | 0.276 | 0.063 |
| Colony 1, Replicate 2 | -0.041 | -0.308 | -3.826 | -0.317 | -0.026 | -2.280 | -6.657 | -0.240 |
| Colony 1, Replicate 3 | 0.010 | 0.050 | 0.209 | 0.093 | 0.009 | 0.275 | 0.426 | 0.071 |
| Colony 1, Replicate 4 | 0.016 | 0.049 | 0.208 | -0.023 | 0.008 | 0.231 | 0.400 | 0.057 |
| Colony 2, Replicate 1 | 0.015 | 0.039 | 0.251 | 0.080 | 0.003 | 0.204 | 0.390 | 0.053 |
| Colony 2, Replicate 2 | 0.026 | 0.047 | 0.247 | 0.105 | 0.038 | 0.236 | 0.444 | 0.060 |
| Colony 2, Replicate 3 | 0.019 | 0.041 | 0.293 | 0.099 | 0.014 | 0.273 | 0.286 | 0.055 |
| Colony 2, Replicate 4 | 0.020 | 0.039 | 0.298 | 0.101 | 0.014 | 0.301 | 0.427 | 0.067 |
µM Fluorescein / OD, Hour 6
| Hour 6: | Neg. Control | Pos. Control | Device 1 | Device 2 | Device 3 | Device 4 | Device 5 | Device 6 |
| Colony 1, Replicate 1 | 0.005 | 0.085 | 0.025 | 0.170 | 0.012 | 0.277 | 0.232 | 0.070 |
| Colony 1, Replicate 2 | 0.000 | 0.078 | -0.007 | 0.138 | 0.005 | 0.236 | 0.173 | 0.051 |
| Colony 1, Replicate 3 | 0.000 | 0.074 | 0.015 | 0.132 | 0.004 | 0.211 | 0.170 | 0.053 |
| Colony 1, Replicate 4 | -0.003 | 0.073 | 0.019 | 0.108 | 0.004 | 0.181 | 0.135 | 0.052 |
| Colony 2, Replicate 1 | 0.000 | 0.068 | 0.018 | 0.140 | 0.005 | 0.294 | 0.164 | 0.053 |
| Colony 2, Replicate 2 | 0.001 | 0.068 | 0.021 | 0.134 | 0.005 | 0.321 | 0.139 | 0.055 |
| Colony 2, Replicate 3 | -0.001 | 0.058 | 0.195 | 0.142 | 0.003 | 0.323 | 0.153 | 0.057 |
| Colony 2, Replicate 4 | 0.001 | 0.077 | 0.261 | 0.160 | 0.005 | 0.315 | 0.183 | 0.052 |
MEFL/particle, Hour 0
| Hour 0: | Neg. Control | Pos. Control | Device 1 | Device 2 | Device 3 | Device 4 | Device 5 | Device 6 |
| Colony 1, Replicate 1 | 1.87E+03 | 6.84E+03 | 3.73E+04 | 1.18E+04 | -7.15E+00 | 3.31E+04 | 3.85E+04 | 8.85E+03 |
| Colony 1, Replicate 2 | -5.72E+03 | -4.30E+04 | -5.34E+05 | -4.42E+04 | -3.57E+03 | -3.18E+05 | -9.29E+05 | -3.35E+04 |
| Colony 1, Replicate 3 | 1.35E+03 | 7.05E+03 | 2.92E+04 | 1.29E+04 | 1.30E+03 | 3.85E+04 | 5.94E+04 | 9.89E+03 |
| Colony 1, Replicate 4 | 2.22E+03 | 6.78E+03 | 2.90E+04 | -3.17E+03 | 1.13E+03 | 3.23E+04 | 5.59E+04 | 7.94E+03 |
| Colony 2, Replicate 1 | 2.12E+03 | 5.41E+03 | 3.50E+04 | 1.12E+04 | 3.96E+02 | 2.85E+04 | 5.44E+04 | 7.42E+03 |
| Colony 2, Replicate 2 | 3.67E+03 | 6.63E+03 | 3.45E+04 | 1.47E+04 | 5.31E+03 | 3.29E+04 | 6.20E+04 | 8.34E+03 |
| Colony 2, Replicate 3 | 2.59E+03 | 5.70E+03 | 4.08E+04 | 1.38E+04 | 2.00E+03 | 3.81E+04 | 3.99E+04 | 7.65E+03 |
| Colony 2, Replicate 4 | 2.75E+03 | 5.44E+03 | 4.16E+04 | 1.41E+04 | 1.93E+03 | 4.20E+04 | 5.96E+04 | 9.29E+03 |
MEFL/particle, Hour 6
| Hour 6: | Neg. Control | Pos. Control | Device 1 | Device 2 | Device 3 | Device 4 | Device 5 | Device 6 |
| Colony 1, Replicate 1 | 7.39E+02 | 1.19E+04 | 3.49E+03 | 2.37E+04 | 1.61E+03 | 3.87E+04 | 3.24E+04 | 9.83E+03 |
| Colony 1, Replicate 2 | -3.73E+01 | 1.09E+04 | -9.65E+02 | 1.93E+04 | 7.02E+02 | 3.29E+04 | 2.42E+04 | 7.07E+03 |
| Colony 1, Replicate 3 | -9.39E+00 | 1.04E+04 | 2.12E+03 | 1.85E+04 | 5.53E+02 | 2.94E+04 | 2.37E+04 | 7.35E+03 |
| Colony 1, Replicate 4 | -3.95E+02 | 1.02E+04 | 2.66E+03 | 1.51E+04 | 5.25E+02 | 2.52E+04 | 1.89E+04 | 7.21E+03 |
| Colony 2, Replicate 1 | 1.86E+01 | 9.44E+03 | 2.55E+03 | 1.95E+04 | 6.55E+02 | 4.10E+04 | 2.29E+04 | 7.37E+03 |
| Colony 2, Replicate 2 | 1.16E+02 | 9.55E+03 | 2.99E+03 | 1.87E+04 | 6.61E+02 | 4.48E+04 | 1.94E+04 | 7.65E+03 |
| Colony 2, Replicate 3 | -8.64E+01 | 8.05E+03 | 2.72E+04 | 1.98E+04 | 4.85E+02 | 4.51E+04 | 2.13E+04 | 8.01E+03 |
| Colony 2, Replicate 4 | 8.18E+01 | 1.08E+04 | 3.65E+04 | 2.24E+04 | 7.16E+02 | 4.40E+04 | 2.56E+04 | 7.23E+03 |
Net Fluorescein a.u., Hour 0
| Hour 0: | Neg. Control | Pos. Control | Device 1 | Device 2 | Device 3 | Device 4 | Device 5 | Device 6 |
| Colony 1, Replicate 1 | 47.57 | 197.62 | 1151.88 | 335.66 | -0.19 | 932.88 | 1182.18 | 235.14 |
| Colony 1, Replicate 2 | 45.27 | 205.85 | 969.36 | 299.01 | 24.18 | 1049.96 | 1533.16 | 248.96 |
| Colony 1, Replicate 3 | 38.87 | 198.86 | 954.12 | 371.05 | 32.40 | 1110.22 | 1675.72 | 248.03 |
| Colony 1, Replicate 4 | 66.34 | 217.10 | 894.64 | -91.52 | 28.19 | 937.34 | 1640.74 | 221.42 |
| Colony 2, Replicate 1 | 54.45 | 133.90 | 1710.55 | 365.80 | 11.12 | 1043.75 | 1498.95 | 220.47 |
| Colony 2, Replicate 2 | 92.53 | 167.25 | 1673.74 | 466.01 | 134.88 | 1097.84 | 1666.74 | 228.32 |
| Colony 2, Replicate 3 | 64.85 | 142.08 | 1145.22 | 459.22 | 50.27 | 1275.22 | 1264.42 | 210.83 |
| Colony 2, Replicate 4 | 72.10 | 147.33 | 1921.49 | 482.31 | 53.09 | 1483.79 | 1633.69 | 266.66 |
Net Fluorescein a.u., Hour 6
| Hour 6: | Neg. Control | Pos. Control | Device 1 | Device 2 | Device 3 | Device 4 | Device 5 | Device 6 |
| Colony 1, Replicate 1 | 180.94 | 3006.80 | 518.03 | 5856.70 | 399.88 | 9163.40 | 2776.30 | 2406.70 |
| Colony 1, Replicate 2 | -9.18 | 2725.72 | -146.04 | 4813.82 | 174.28 | 7738.72 | 2412.92 | 1727.52 |
| Colony 1, Replicate 3 | -2.33 | 2620.59 | 327.64 | 4636.69 | 138.59 | 6962.19 | 2321.79 | 1784.49 |
| Colony 1, Replicate 4 | -98.09 | 2566.00 | 402.77 | 3805.10 | 131.88 | 6126.20 | 2043.30 | 1757.60 |
| Colony 2, Replicate 1 | 4.52 | 2371.29 | 423.29 | 4889.79 | 160.30 | 9815.69 | 1992.19 | 1813.19 |
| Colony 2, Replicate 2 | 28.22 | 2404.72 | 470.44 | 4746.82 | 162.46 | 10836.42 | 1753.32 | 1934.12 |
| Colony 2, Replicate 3 | -21.15 | 1989.19 | 4440.79 | 5044.49 | 120.23 | 10783.19 | 1788.69 | 1996.19 |
| Colony 2, Replicate 4 | 19.83 | 2750.03 | 5874.63 | 5626.03 | 180.40 | 10822.83 | 2288.53 | 1856.33 |
Net Abs600, Hour 0
| Hour 0: | Neg. Control | Pos. Control | Device 1 | Device 2 | Device 3 | Device 4 | Device 5 | Device 6 |
| Colony 1, Replicate 1 | 0.154 | 0.175 | 0.187 | 0.172 | 0.161 | 0.171 | 0.186 | 0.161 |
| Colony 1, Replicate 2 | -0.048 | -0.029 | -0.011 | -0.041 | -0.041 | -0.020 | -0.010 | -0.045 |
| Colony 1, Replicate 3 | 0.174 | 0.171 | 0.198 | 0.174 | 0.151 | 0.175 | 0.171 | 0.152 |
| Colony 1, Replicate 4 | 0.181 | 0.194 | 0.187 | 0.175 | 0.151 | 0.176 | 0.178 | 0.169 |
| Colony 2, Replicate 1 | 0.156 | 0.150 | 0.296 | 0.198 | 0.170 | 0.222 | 0.167 | 0.180 |
| Colony 2, Replicate 2 | 0.153 | 0.153 | 0.294 | 0.192 | 0.154 | 0.202 | 0.163 | 0.166 |
| Colony 2, Replicate 3 | 0.152 | 0.151 | 0.170 | 0.202 | 0.152 | 0.203 | 0.192 | 0.167 |
| Colony 2, Replicate 4 | 0.159 | 0.164 | 0.280 | 0.208 | 0.167 | 0.214 | 0.166 | 0.174 |
Net Abs600, Hour 6
| Hour 6: | Neg. Control | Pos. Control | Device 1 | Device 2 | Device 3 | Device 4 | Device 5 | Device 6 |
| Colony 1, Replicate 1 | 1.484 | 1.535 | 0.899 | 1.496 | 1.507 | 1.434 | 0.519 | 1.484 |
| Colony 1, Replicate 2 | 1.493 | 1.516 | 0.917 | 1.512 | 1.504 | 1.424 | 0.604 | 1.481 |
| Colony 1, Replicate 3 | 1.504 | 1.531 | 0.938 | 1.521 | 1.520 | 1.433 | 0.594 | 1.471 |
| Colony 1, Replicate 4 | 1.505 | 1.528 | 0.917 | 1.524 | 1.523 | 1.471 | 0.657 | 1.477 |
| Colony 2, Replicate 1 | 1.469 | 1.523 | 1.006 | 1.517 | 1.484 | 1.452 | 0.528 | 1.492 |
| Colony 2, Replicate 2 | 1.469 | 1.526 | 0.954 | 1.537 | 1.490 | 1.465 | 0.547 | 1.532 |
| Colony 2, Replicate 3 | 1.484 | 1.497 | 0.990 | 1.547 | 1.502 | 1.449 | 0.509 | 1.511 |
| Colony 2, Replicate 4 | 1.469 | 1.543 | 0.976 | 1.523 | 1.528 | 1.492 | 0.542 | 1.556 |
Colony Forming Units
Two colonies each of negative and positive control were grown overnight and diluted to 0.1 OD600 in triplicate the next morning. This resulted in 12 total starting samples: 3 for each colony, 6 for each control strain. Then, three 1:20 dilutions were done on each sample, followed by two 1:10 dilutions. These last three were plated. Rather than plate 100 µL on one plate, all samples had 33 µL plated on one-third of a plate. A diagram of this experiment is below:
After these plates were allowed to grow at 30 degrees Celsius overnight, the three team members of BostonU counted all of the colonies on each of the plates. These numbers, as well as the calculated number of Colony-Forming Units per 0.1 OD600 from each replicate, can be found in the tables below.
Data
Negative Control, Colony A
| Replicate | 8 x 10^4 dilution | 8 x 10^5 dilution | 8 x 10^6 dilution | Colony Forming Units |
| Replicate 1 | 703 | 522 | 427 | 3416000000 |
| Replicate 2 | 688 | 875 | 723 | 55040000 |
| Replicate 3 | 824 | 776 | 506 | 4048000000 |
Negative Control, Colony B
| Replicate | 8 x 10^4 dilution | 8 x 10^5 dilution | 8 x 10^6 dilution | Colony Forming Units |
| Replicate 1 | 447 | 407 | 334 | 2672000000 |
| Replicate 2 | 416 | 382 | 253 | 2024000000 |
| Replicate 3 | 444 | 396 | 182 | 1456000000 |
Positive Control, Colony A
| Replicate | 8 x 10^4 dilution | 8 x 10^5 dilution | 8 x 10^6 dilution | Colony Forming Units |
| Replicate 1 | 623 | 396 | 499 | 316800000 |
| Replicate 2 | 801 | 272 | 637 | 217600000 |
| Replicate 3 | 625 | 453 | 611 | 362400000 |
Positive Control, Colony B
| Replicate | 8 x 10^4 dilution | 8 x 10^5 dilution | 8 x 10^6 dilution | Colony Forming Units |
| Replicate 1 | 843 | 499 | 366 | 2928000000 |
| Replicate 2 | 452 | 637 | 424 | 3392000000 |
| Replicate 3 | 510 | 611 | 274 | 2192000000 |