INTERLAB STUDY
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 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 grown c 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
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 |
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 OD 600 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 | Arith. 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 Abs 600 measurements to an estimated number of cells.
CALIBRATION 3: FLUORESCENCE 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
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, R3 | |||||||||
lolita
Alberto Conejero lerolerolero
I am member of the Instituto Universitario de Matemática Pura y Aplicada of the UPV. I am also interested in Biomedical Data Analysis, Graph Theory, Network Science, and in the applications of Mathematics to Computational, Systems and Synthetic Biology, and Communication Networks.
I am the author of more than 50 research articles published in international research journals. In addition, I have stayed at the following universities for short periods: in Bowling Green (OH) and Kent (OH) (USA), Lecce (Italy), Prague (Czech Rep.) And Tübingen (Germany).
Before being Director of the Department of Applied Mathematics, I held the position of Director of Academic Performance and Curricular Students Assessment Area of the Vice-rectorate of Students and Culture of the UPV. Previously I held these positions university: Deputy Dean of the ETSINF (formerly Faculty of Informatics) (2004-2009), and Secretary of the Commission of the Strategic Plan of the UPV for the period 2007-2014 (2005-2007).
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