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Revision as of 01:07, 18 October 2018

iGEM ECUADOR
C-lastin, Interlab
INTERLAB
What did we do?
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
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


length: 918 bp


length: 918 bp


length: 918 bp


length: 918 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.
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