Revision as of 22:26, 3 October 2018

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

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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

4. Measurement of Abs600 and Fluorescence of Transformed Cell Samples

Raw Plate Readings for Abs600 and Fluorescence

Fluorescence per OD

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