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
