Difference between revisions of "Team:UIUC Illinois/Interlab"

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<div style="text-align:center; color: black; font-size:3vw">InterLab Study</div>
 
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<p style="color: black; font-size:1.2vw;margin-left: 5vw; align=left; margin-right:5vw">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 m1000 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 CL-X, (3) graphing a particle standard curve for monodisperse silica microspheres and a Fluorescein standard curve (4) measuring the GFP fluorescence and absorbance of samples of previously transformed DH5α cells taken over 2 hour intervals and 5) counting colony forming units (CFUs) per 0.1 OD600 E. coli cultures. </p>
 
<p style="color: black; font-size:1.2vw;margin-left: 5vw; align=left; margin-right:5vw">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 m1000 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 CL-X, (3) graphing a particle standard curve for monodisperse silica microspheres and a Fluorescein standard curve (4) measuring the GFP fluorescence and absorbance of samples of previously transformed DH5α cells taken over 2 hour intervals and 5) counting colony forming units (CFUs) per 0.1 OD600 E. coli cultures. </p>
  
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Revision as of 19:14, 18 September 2018

InterLab Study

As a team, we took part in iGEM’s Fifth International Interlaboratory Study in synthetic biology. Difficulty in taking reliable and reproducible measurements remains a key obstacle in the field of synthetic biology, especially for fluorescence data. Data from different groups usually cannot be compared because they are reported in different units or processed in different ways. The goal of the iGEM InterLab Study is to identify and correct the sources of systematic variability in synthetic biology measurements.

For the fifth installment of the InterLab, iGEM wants to answer the following question: Can we reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of OD? The parts used include six test devices (BBa_J364000, BBa_J364001, BBa_J364002, BBa_J364003, BBa_J364004, BBa_J364005), as well as a positive (BBa_I20270) and negative (BBa_R0040) control. All parts are located in the pSB1C3 plasmid and carry chloramphenicol resistance.

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 m1000 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 CL-X, (3) graphing a particle standard curve for monodisperse silica microspheres and a Fluorescein standard curve (4) measuring the GFP fluorescence and absorbance of samples of previously transformed DH5α cells taken over 2 hour intervals and 5) counting colony forming units (CFUs) per 0.1 OD600 E. coli cultures.

OD600 Reference Point of LUDOX CL-X

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

LUDOX-HS40 Water
Replicate 1 0.058 0.037
Replicate 2 0.058 0.036
Replicate 3 0.058 0.036
Replicate 4 0.058 0.036
Mean 0.058 0.036
Corrected Abs600 0.022
Reference OD600 0.063
OD600/Abs600 2.870

Particle and Fluorescein standard curve

Table Data for the Fluorescein Standard Curve

μM Fluorescein 10.00 5 2.5 1.25 .625 .313 .156 0.078 0.039 0.0195 0.0098 0
Replicate 1 40576 16965 8970 4673 2366 1175 596 295 150 73 38 1
Replicate 2 40733 17202 8941 4671 2353 1184 533 323 150 76 37 1
Replicate 3 40610 16977 8670 4549 2024 1129 653 363 230 79 54 1
Replicate 4 40996 14056 9841 5352 3835 1230 886 490 311 108 74 1
Arithmetic Mean 40730 16300 9106 4811 2645 1180 667.0 367.8 210.3 84.00 50.75 1.000
Arithmetic Standard Deviation 190.5 1500 508.6 365.1 809.3 41.40 154.0 86.14 77.03 16.19 17.35 0
Arithmetic Net Mean 40730 16300 9105 4810 2644 1179 666.0 366.8 209.3 83.00 49.75
]
μM Fluorescein 10.00 5.00 2.50 1.25 0.63 0.31 0.16 0.08 0.04 0.02 0.01
μM Fluorescein/a.u. 0.000246 0.000307 0.000275 0.00026 0.000236 0.000265 0.000235 0.000213 0.000187 0.000235 0.000196
Mean uM Fluorescein/a.u. .000269
Mean uM Fluorescein/a.u. 1.62E+09

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