Team:IISER-Mohali/InterLab
Interlab
Replicating the same measurements in different laboratories becomes a challenging task in Synthetic Biology. Considering the case of Fluorescence data, this year iGEM’s Measurement Committee aims to develop absolute units for measurements of green fluorescent protein (GFP) in a plate reader and to see the possibility of reducing lab-to-lab variability in fluorescence measurements by normalizing to colony-forming units (CFUs) instead of optical density (OD). For that purpose, we were required to measure the cell density of Escherichia coli (E. coli) DH5⍺ cells using two different methods. This means teams have to use the same exact protocol around the world to produce common, comparable units for measuring GFP with different plate readers. For more information on the protocol, check out iGEM's official Interlab Study webpage. Below is the data that we collected for the Interlab Study:
OD600 Reference Point
| LUDOX-HS40 | H2O | |
|---|---|---|
| Replicate 1 | 0.064 | 0.030 |
| Replicate 2 | 0.055 | 0.031 |
| Replicate 3 | 0.071 | 0.031 |
| Replicate 4 | 0.062 | 0.029 |
| Arith. Mean | 0.063 | 0.030 |
| Corrected Abs600 | 0.033 | |
| Reference OD600 | 0.063 | |
| OD600/Abs600 | 1.924 |
Plate Pattern
| A1 | A2 | A3 | A4 | A5 | A6 | A7 | A8 | A9 |
| B1 | B2 | B3 | B4 | B5 | B6 | B7 | B8 | B9 |
| C1 | C2 | C3 | C4 | C5 | C6 | C7 | C8 | C9 |
| D1 | D2 | D3 | D4 | D5 | D6 | D7 | D8 | D9 |
| E1 | E2 | E3 | E4 | E5 | E6 | E7 | E8 | E9 |
| F1 | F2 | F3 | F4 | F5 | F6 | F7 | F8 | F9 |
| G1 | G2 | G3 | G4 | G5 | G6 | G7 | G8 | G9 |
| H1 | H2 | H3 | H4 | H5 | H6 | H7 | H8 | H9 |
Results and Discussion
Flourescence Readings at different cell densities
In order to estimate the actual amount of cells in fluorescence measurement, silica beads provided in the iGEM kit were used, which are modeled according to a typical E. coli cell and so the beads were expected to scatter light in a similar way to E. coli cells. The cell density of a particular sample could be then converted to an equivalent concentration of beads and becomes a more accountable and universal method for measurement.
Graph showing the comparison of the fluoroscence readings at T = 0 h for each provide device
Graph showing the comparison of the fluoroscence readings at T = 6 h for each device
Abs600 Raw Readings
Graph showing the comparison of the net Abs600 at T = 0 h for each device
Graph showing the comparison of the net Abs600 at T = 6 h for each device
CFU Data
Plating known volume of sample could provide the approximation for cell concentration. The cell concentration in the sample is thought to be directly proportional to the number of CFUs. Using a scaling factor computed from negative and positive control CFUs, a conversion factor from absorbance to CFU can be computed.
| Culture | Replicate | Dilution 3 | Dilution 4 | Dilution 5 | CFU/mL |
|---|---|---|---|---|---|
| Positive Control (Culture1) | Replicate 1 | 532 | 215 | 39 | 1.72*1009 |
| Positive Control (Culture1) | Replicate 2 | 387 | 232 | 87 | 1.86*1009 |
| Positive Control (Culture1) | Replicate 3 | 519 | 266 | 45 | 2.13*1009 |
| Positive Control (Culture2) | Replicate 1 | TNTC | 178 | 61 | 1.42*1009 |
| Positive Control (Culture2) | Replicate 2 | TNTC | 261 | 68 | 2.09*1009 |
| Positive Control (Culture2) | Replicate 3 | TNTC | 206 | 94 | 1.65*1009 |
| Negative Control (Culture1) | Replicate 1 | 416 | 204 | 106 | 1.63*1009 |
| Negative Control (Culture1) | Replicate 2 | 378 | 261 | 43 | 2.09*1009 |
| Negative Control (Culture1) | Replicate 3 | 394 | 186 | 66 | 1.49*1009 |
| Negative Control (Culture2) | Replicate 1 | TNTC | 239 | 52 | 1.91*1009 |
| Negative Control (Culture2) | Replicate 2 | TNTC | 217 | 63 | 1.74*1009 |
| Negative Control (Culture2) | Replicate 3 | TNTC | 238 | 79 | 1.90*1009 |
Counting colony-forming units (CFUs) from the sample