Team:Valencia UPV/InterLab

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Index

Introduction
What is this year's goal?
Plate Reader Setup
Used Parts
Calibration 1: OD₆₀₀ Reference Point
Calibration 2: Particle Standard Curve
Calibration 3: Fluorescence Standard Curve
Experiment
Application

Introduction

Do you imagine doing an experiment that could not be repeated? What if, after performing the same experiment several times, you obtain different results each time? This is a <6>common problem throughout almost all laboratories in the entire world. A challenge, not just for Synthetic Biology but for any type of science, is taking reliable and repeatable measurements.

Over the past four years, the iGEM Measurement Committee has been developing a series of experiments to make the biggest interlaboratory studies ever done in synthetic biology, and, in that way, try to fix all possible variables within a particular protocol.

What is this year's goal?

To know if there is any chance to reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or c-forming units (CFUs) instead of optical density (OD).

In order to compute the cell count in our samples, we will use two orthogonal approaches:

Approach 1: Converting between absorbance of cells to absorbance of a known concentration of beads

The theory under how absorbance is measured is quite simple: a liquid sample of cells scatter light in a way or another depending on the number of cells this sample contains. The Committee provides us a sample with silica beads which are almost the same size and shape as a typical E. coli cell. So, when mixed with water, we obtain a liquid that should scatter light in a similar way as our E. coli sample does.

Because we know the concentration of beads, the absorbance measurement from a particular cell sample could be converted into an “equivalent concentration of beads” measurement, so that they are more universal and comparable measurements between different labs.

Approach 2: Counting c-forming units (CFUs) from the sample

This method relies on the idea that every colony grown in our plate comes from a single cell. So, if we spread a known cell culture volume over an agar plate and then we count the number of colonies, we should have an idea on how many cells our liquid sample had. We will have to determine the number of CFUs in positive and negative control samples in order to compute a conversion factor from absorbance to CFU.

Plate reader setup

Absorbance₆₀₀

Absorbance Endpoint

Full Plate

Wavelengths: 600 nm

Read Speed: Normal, Delay: 100 msec, Measurements/Data Point: 8

Fluorescence

Excitation: 485, Emission: 528

Optics: Top, Gain: 50

Light Source: Xenon Flash, Lamp Energy: High

Read Speed: Normal, Delay: 100 msec, Measurements/Data Point: 10

Read Height: 7 mm

Used Parts

Device	Part number	Plate	Location
Negative control	BBa_R0040	Kit Plate 7	Well 2D
Positive control	BBa_I20270	Kit Plate 7	Well 2B
Test Device 1	BBa_J364000	Kit Plate 7	Well 2F
Test Device 2	BBa_J364001	Kit Plate 7	Well 2H
Test Device 3	BBa_J364002	Kit Plate 7	Well 2J
Test Device 4	BBa_J364007	Kit Plate 7	Well 2L
Test Device 5	BBa_J364008	Kit Plate 7	Well 2N
Test Device 6	BBa_J364009	Kit Plate 7	Well 2P

Table 1. Used parts, links, and well information.

Calibration 1: OD₆₀₀ Reference Point

Using LUDOX CL-X as a point reference to obtain a conversion factor to transform our absorbance (Abs₆₀₀) data from our plate reader into a comparable OD₆₀₀ measurement as would be obtained in a spectrophotometer.

	LUDOX CL-X	H2O
R1	0.061	0.051
R2	0.060	0.049
R3	0.061	0.043
R4	0.062	0.049
Arithmetic Mean	0.061	0.048
Corrected Abs₆₀₀	0.013
Reference OD₆₀₀	0.063
OD₆₀₀/Abs₆₀₀	4.846

Table 2. OD₆₀₀ reference point.

Calibration 2: Particle Standard Curve

This allows us to construct a standard curve of particle concentration which can be used to convert Abs₆₀₀ measurements to an estimated number of cells.

Figure 1. Particle standard curve and particle standard curve in logarithmic scale.

Calibration 3: Fuorescence Standard Curve

Absolute fluorescence values cannot be directly compared from one instrument to another. In order to compare fluorescence output of test devices between teams, it is necessary for each team to create a standard fluorescence curve.

Figure 2. Fluorescein standard curve and fluorescein standard curve in logarithmic scale.

Experiment

Fluorescence Raw Readings:

Hour 0:	Neg. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6	LB+Chlor (blank)
C1, R1	3618	3087	5355	2950	3402	4799	4112	3901	3350
C1, R2	3648	3414	5158	2887	3260	4647	4159	3758	3193
C1, R3	3273	3442	5077	1611	3331	4607	3972	3793	3234
C1, R4	3301	1381	5307	1074	3446	4519	4416	3804	3221
C2, R1	3350	3537	5091	3702	3976	4621	4517	3969	3256
C2, R2	3262	3409	4758	3623	3660	4208	4679	3867	3265
C2, R3	3255	3401	4784	3620	3662	4248	4481	3951	3231
C2, R4	3225	3020	4855	3518	3672	4451	4281	4183	4070

Table 3. Fluorescence raw readings, 0 hours.

Hour 6:	Neg. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6	LB+Chlor (blank)
C1, R1	4087	23103	44980	7751	4312	29208	6183	10720	3306
C1, R2	4238	23435	46319	7594	4341	28505	6522	10895	3177
C1, R3	4194	23871	45601	7757	4371	28765	6359	10515	3260
C1, R4	4195	24568	45744	7709	4546	29372	6156	11334	3205
C2, R1	4398	11707	43194	17428	4756	28009	11866	10612	3269
C2, R2	4351	12443	43496	17570	4804	28545	11928	10545	3250
C2, R3	4291	11451	43156	17421	4796	28475	11726	10544	3295
C2, R4	4292	12396	42704	17353	4938	28869	11517	10546	3231

Table 4. Fluorescence raw readings, 6 hours.

Abs₆₀₀ Raw Readings:

Hour 0:	Neg. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6	LB+Chlor (blank)
C1, R1	0.067	0.055	0.060	0.056	0.059	0.059	0.057	0.059	0.047
C1, R2	0.061	0.056	0.058	0.054	0.059	0.056	0.058	0.058	0.046
C1, R3	0.057	0.058	0.057	0.047	0.065	0.058	0.056	0.059	0.045
C1, R4	0.062	0.047	0.058	0.048	0.058	0.057	0.057	0.059	0.045
C2, R1	0.055	0.055	0.058	0.055	0.059	0.055	0.056	0.056	0.046
C2, R2	0.055	0.059	0.054	0.056	0.057	0.054	0.059	0.058	0.047
C2, R3	0.071	0.055	0.056	0.071	0.056	0.057	0.057	0.058	0.046
C2, R4	0.056	0.059	0.055	0.057	0.055	0.059	0.056	0.059	0.049

Table 5. Abs₆₀₀ Raw Readings, 0 hours.

Hour 6:	Neg. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6	LB+Chlor (blank)
C1, R1	0.595	0.513	0.471	0.391	0.554	0.518	0.083	0.487	0.066
C1, R2	0.567	0.527	0.508	0.363	0.556	0.519	0.084	0.501	0.053
C1, R3	0.566	0.524	0.479	0.381	0.566	0.501	0.083	0.490	0.059
C1, R4	0.580	0.570	0.530	0.373	0.549	0.559	0.088	0.551	0.051
C2, R1	0.611	0.513	0.519	0.531	0.515	0.530	0.117	0.520	0.053
C2, R2	0.562	0.538	0.523	0.536	0.522	0.543	0.116	0.517	0.057
C2, R3	0.561	0.493	0.517	0.507	0.518	0.502	0.106	0.488	0.054
C2, R4	0.555	0.537	0.498	0.512	0.533	0.508	0.101	0.491	0.06

Table 6. Abs₆₀₀ Raw Readings, 6 hours.

μM Fluorescein/OD:

Hour 0:	Neg. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6
C1, R1	0.415	-1.019	4.780	-1.377	0.134	3.742	2.362	1.423
C1, R2	0.940	0.685	5.075	-1.185	0.160	4.506	2.495	1.459
C1, R3	0.101	0.496	4.760	-25.149	0.150	3.273	2.079	1.237
C1, R4	0.155	-28.512	4.973	-22.179	0.536	3.352	3.086	1.291
C2, R1	0.324	0.968	4.739	1.536	1.716	4.700	3.908	2.210
C2, R2	-0.012	0.372	6.610	1.233	1.224	4.175	3.652	1.696
C2, R3	0.030	0.585	4.813	0.482	1.336	2.865	3.522	1.859
C2, R4	-3.741	-3.254	4.055	-2.138	-2.056	1.581	0.934	0.350

Table 7. μM Fluorescein/OD, 0 hours.

Hour 6:	Neg. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6
C1, R1	0.046	1.373	3.189	0.424	0.064	1.776	5.245	0.546
C1, R2	0.064	1.325	2.939	0.442	0.072	1.684	3.344	0.534
C1, R3	0.057	1.374	3.124	0.433	0.068	1.788	4.002	0.522
C1, R4	0.058	1.276	2.752	0.433	0.083	1.596	2.472	0.504
C2, R1	0.063	0.568	2.655	0.918	0.100	1.607	4.163	0.487
C2, R2	0.068	0.592	2.677	0.927	0.104	1.613	4.558	0.491
C2, R3	0.061	0.576	2.668	0.966	0.100	1.742	5.025	0.518
C2, R4	0.066	0.595	2.793	0.968	0.112	1.764	6.263	0.526

Table 8. μM Fluorescein/OD, 6 hours.

MEFL/particle:

Hour 0:	Neg. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6
C1, R1	1.31E+05	-3.22E+05	1.51E+06	-4.35E+05	4.24E+04	1.18E+06	7.46E+05	4.50E+05
C1, R2	2.97E+05	2.16E+05	1.60E+06	-3.75E+05	5.05E+04	1.42E+06	7.89E+05	4.61E+05
C1, R3	3.18E+04	1.57E+05	1.50E+06	-7.95E+06	4.75E+04	1.03E+06	6.57E+05	3.91E+05
C1, R4	4.90E+04	-9011826	1.57E+06	-7.01E+06	1.70E+05	1.06E+06	9.75E+05	4.08E+05
C2, R1	1.02E+05	3.06E+05	1.50E+06	4.85E+05	5.43E+05	1.49E+06	1.24E+06	6.98E+05
C2, R2	-3.67E+03	1.18E+05	2.09E+06	3.90E+05	3.87E+05	1.32E+06	1.15E+06	5.36E+05
C2, R3	9.40E+03	1.85E+05	2.09E+06	1.52E+05	4.22E+05	9.06E+05	1.11E+06	5.88E+05
C2, R4	-1.18E+06	-1.03E+06	1.28E+06	-6.76E+05	-6.50E+05	3.73E+05	2.95E+05	1.11E+05

Table 9. MEFL/particle, 0 hours.

Hour 6:	Neg. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6
C1, R1	1.45E+04	4.34E+05	1.01E+06	1.34E+05	2.02E+04	5.61E+05	1.66E+06	1.73E+05
C1, R2	2.02E+04	4.19E+05	9.29E+05	1.40E+05	2.27E+04	5.32E+05	1.06E+06	1.69E+05
C1, R3	1.80E+04	4.34E+05	9.87E+05	1.37E+05	2.15E+04	5.65E+05	1.26E+06	1.65E+05
C1, R4	1.83E+04	4.03E+05	8.70E+05	1.37E+05	2.64E+04	5.05E+05	7.81E+05	1.59E+05
C2, R1	1.98E+04	1.80E+05	8.39E+05	2.90E+05	3.15E+04	5.08E+05	1.32E+06	1.54E+05
C2, R2	2.14E+04	1.87E+05	8.46E+05	2.93E+05	3.27E+04	5.10E+05	1.44E+06	1.55E+05
C2, R3	1.92E+04	1.82E+05	8.43E+0.5	3.05E+05	3.17E+04	5.51E+05	1.59E+06	1.64E+05
C2, R4	2.10E+04	1.88E+05	8.83E+05	3.06E+05	3.54E+04	5.61E+05	1.98E+06	1.66E+05