Team:Ecuador/Interlab

We tried to reduce the variability in the measurement of GFP expression, for this reason we used a direct method to determine the mean expression level of GFP per cel so we can standardize a universal unit to measure the absorbance, the “equivalent concentration of beads”.

On the other hand we tried to determine a simple way to count the number of present cells in a sample, transforming the absorbance to CFU.

How did we do it?

In order to avoid distortions in the results due to differences in the experimentation processes, we follow the procedures described in the iGEM protocol (Plate reader protocol, Competent cells, Transformation)l

Calibration

Before starting the experiments, we performed the OD600 calibration measurements following the LUDOX protocol, the particle Estandar Curve with the Microsphere Protocol, and the fluorescence standard curve with the Fluorescein Protocol. This allowed us to familiarize ourselves with the plate reader required for subsequent measurements. The plate reader that we used had the following characteristics:
Label: BioTek / Model: Cytation 5 / Emission: 528 / Excitation: 485 / Optic position: Top / Temperature: 25 ºC / Shake: 425 cpm for 30 sec before every measurement.

Cell measurement

We started by transforming E. coli DH5 alpha with the plasmid described in the protocol, and after incubation and growth in LB-medium, we diluted the culture to Abs600 0.2 and proceeded with the samples.

Colony units per 0.1 OD600 E. coli cultures

In order to determine the cell concentration of the culture we followed the CFU protocol that basically consisted in diluting a culture to certain absorbance, measure the OD600 that has to be in a value of 0.1, and make serial dilutions to later count the colonies and calculate the CFU/mL.

BioBricks

Device	Part Number
Negative control	BBa_R0040
Positive control	BBa_I20270
Test Device 1	BBa_J364000
Test Device 2	BBa_J364001
Test Device 3	BBa_J364002
Test Device 4	BBa_J364007
Test Device 5	BBa_J364008
Test Device 6	BBa_J364009

Structure
(Point with the cursor on the device)

length: 54 bp

length: 919 bp

length: 918 bp

Results

Particle standard curves

Fluorescein standard curves

Converting between absorbance of cells to absorbance of a known concentration of beads

Counting colony-forming units (CFUs) from the sample

EXPERIENCE

The interlab protocols were easy to understand and simple. We recommend the following:

Before each measurement, shake the plates in the equipment since the particles tend to precipitate in the well.
If the iGEM equipment does not have the plate reader in the same place as your laboratory, they must take care of their samples in continuous refrigeration during transport and carry out the activities with time.

We learned many things in the interlab challenge: the protocols, the management of the plate reader and how it can be used in later processes of our project. It also helped us strengthen cooperation with other universities.

ACKNOWLEDGEMENT

As a team, we would like to thank the Biomedicine team of Universidad Tecnica Equinoccial and especially Linda Guaman, member of their research team, for allowing us to use their measurement equipment, which were essential for the completion of the Interlab.