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