Team:Valencia UPV/InterLab

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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.

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

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

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

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.

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.

Experiment

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.
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
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
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

Application

Thanks to the InterLab Study initiative, the Valencia UPV iGEM team has decided to go one step further in this competition. From Interlab experimental data, we know that we can obtain equivalence factors between arbitrary fluorescence units and equivalent molecules of fluorophore (MEFL), such as fluorescein. However, for what reason is this conversion useful?

In Printeria, one of the most important tools for characterization has been modeling. We have developed mathematical models of genetic constructions and we have characterized the parts through parameter optimization. Thanks to the conversion of experimental fluorescence data to MEFL, we have been able to obtain more realistic parameters values and, therefore, closer to reality. If you want to discover our results, visit our Modeling section.