Team:WashU StLouis/InterLab

InterLab Study

As a team, we decided to participate in iGEM's Fourth International Interlaboratory Study along with other teams from around the world. This study is organized by iGEM's measurement committee in an effort to better measurement techniques in the field of synthetic biology. Our involvement in the study required that we submit measurement data dealing with the fluorescence of GFP and OD associated with cells transformed with different test devices. Throughout our experiments, we tested 8 plasmids (2 controls and 6 test devices), and we measured the absorbance and fluorescence of our samples using a Tecan Infinite M200 Pro plate reader. We broke the given procedure into four main components: (1) transforming the two controls and 6 test devices into competent DH5α cells, (2) measuring the OD600 reference point of LUDOX-HS40, (3) graphing a standard curve for Fluorescein, and (4) measuring the GFP fluorescence and absorbance of samples of previously transformed DH5α cells taken over 2 hour intervals.

1. Transformation of Test Devices and Controls

During this part of the interlab study, we transformed all of the control plasmids and test device plasmids into DH5α E. coli cells. After transforming the cells successfully and growing them on plates overnight, we picked two colonies from each of the plates and prepared overnight cultures for them.

Plates of DH5α Cells After Transformation

Top (from left to right): Postive control (BBa_I20270), Negative control (BBa_R0040), Test device 1 (BBa_J364000), Test device 2 (BBa_J364001) Bottom (from left to right): Test device 3 (BBa_J364002), Test device 4 (BBa_J364003), Test device 5 (BBa_J364004), Test device 6 (BBa_J364005)

2. Measurement of LUDOX-HS40 OD600 Reference Point

Abs600 and OD600 Data/Calculations for LUDOX-HS40 and Water

LUDOX-HS40 Water
Replicate 1 0.095 0.0893
Replicate 2 0.0923 0.0833
Replicate 3 0.0943 0.0866
Replicate 4 0.1016 0.0859
Mean 0.0958 0.087525
Corrected Abs600 0.008275
Reference OD600 0.0425
OD600/Abs600 5.1359517

3. Graphing a Standard Curve for Fluorescein

Table Data for the Fluorescein Standard Curve

μM Fluorescein 50.00 25 12.5 6.25 3.125 1.5625 0.78125 0.390625 0.195313 0.097656 0.048828 0
Replicate 1 39439 25542 15476 8609 4655 2432 1205 666 359 193 96 2
Replicate 2 40342 26003 15496 8750 4671 2457 1268 694 366 202 102 2
Replicate 3 39940 25812 15591 8808 4784 2567 1295 726 386 213 107 2
Replicate 4 39680 25076 15743 9165 4973 2680 1300 741 410 211 119 5
Arithmetic Mean 39850.25 25608.25 15576.5 8833 4770.75 2531.75 1267 706.75 380.25 204.75 106 2.75
Arithmetic Standard Deviation 386.4301 402.0865 121.81 236.5826 146.5478 116.3826 43.65776 33.5 22.89651 9.17878 9.763879 1.5
μM Fluorescein 50.00 25 12.5 6.25 3.125 1.5625 0.78125 0.390625 0.195313 0.097656 0.048828
Mean μM Fluorescein/a.u. 0.001255 0.000976 0.000802 0.000708 0.000655 0.000617 0.000617 0.000553 0.000514 0.000477 0.000461
Mean of med-high levels = 0.000752

4. Measurement of Abs600 and Fluorescence of Transformed Cell Samples

Raw Plate Readings for Abs600 and Fluorescence

Adjusted Abs600 and Fluorescence Measurements

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