Difference between revisions of "Team:Ecuador/Interlab"
Tefiducky8 (Talk | contribs) |
Tefiducky8 (Talk | contribs) |
||
| Line 199: | Line 199: | ||
<h3>Index</h3> | <h3>Index</h3> | ||
<ul> | <ul> | ||
| − | <li><a href="#WWD">What did we do?</a></li> | + | <li><a href="#WWD" style="text-decoration: none; color:black;">What did we do?</a></li> |
| − | <li><a href="#HWD">How did we do it?</a></li> | + | <li><a href="#HWD" style="text-decoration: none; color:black;">How did we do it?</a></li> |
| − | <li><a href="#BIO">BioBricks</a></li> | + | <li><a href="#BIO" style="text-decoration: none; color:black;">BioBricks</a></li> |
| − | <li><a href="#RES">Results</a></li> | + | <li><a href="#RES" style="text-decoration: none; color:black;">Results</a></li> |
| − | <li><a href="#EXP">Experience</a></li> | + | <li><a href="#EXP" style="text-decoration: none; color:black;">Experience</a></li> |
| − | <li><a href="#ACK">Acknowledgement</a></li> | + | <li><a href="#ACK" style="text-decoration: none; color:black;">Acknowledgement</a></li> |
</ul> | </ul> | ||
</div> | </div> | ||
Latest revision as of 02:43, 19 November 2018
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 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.
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
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 |
(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.