Difference between revisions of "Team:Valencia UPV/InterLab"

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

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.

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:

Table 3. Fluorescence raw readings, 0 hours.

Table 4. Fluorescence raw readings, 6 hours.

Abs₆₀₀ Raw Readings:

Table 5. Abs₆₀₀ Raw Readings, 0 hours.

Table 6. Abs₆₀₀ Raw Readings, 6 hours.

μM Fluorescein/OD:

Table 8. μM Fluorescein/OD, 6 hours.

MEFL/particle:

Table 10. MEFL/particle, 6 hours.