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Team:Tec-Monterrey/antifreeze/results
From 2012.igem.org
ANTIFREEZE
To prove the activity of scFv IgE+GFP, we needed to detect both parts separately. To de GFP emission we designed the following experiment.
Using a 96-well fluorescence plate (a black one) we filled some wells with supernatant from our induced Pichia pastoris with scFv IgE+GFP, some with the intracellular extract from the same culture, and the same treatments but for our P.pastoris with scFv His. The fluorescence plate for the experiment was designed as follows:
Dem future works
Experiment 1
The standardization of the protocol for testing the viability of cells producing antifreeze proteins was based on the article "Evaluation of tolerance for cryopreservation of two strains of Escherichia coli K12 often used in biotechnology" and iGEM Amsterdam 2011’s protocol.
1) Three strains were tested TOP10, JM109, and BL21. Using strains transformed with AFP and untransformed (as control)
2) The strains were placed at -20 ° C for 24 hours
Tec-Monterrey
iGEM 2012
3) Thaw for 2 hours on ice
4) Inoculated in plates in a dilution of 10 ^ -5 (cycle 0)
5) The cells were frozen for 2.5 hours (-20 °C)
6) Afterwards they were placed 20 min on ice and inoculated in a dilution of 10 ^ -5 (cycle 1)
7) The cells were frozen for 1 hour
8) Thaw for 20 minutes on ice and inoculated in a dilution of 10 ^ -5 (cycle 2)
9) Freeze for 1 hour
10) Thaw for 20 minutes on ice and inoculated in a dilution of 10 ^ -5 (cycle 3)
11) Freeze for 1 hour
12) Thaw for 20 minutes on ice inoculated in a dilution of 10 ^ -5 (Cycle 4)
13) Incubate at 37 ° C
Results
The plates resulted countless so the protocol needed to be restructured
Experiment 2
The protocol was re-standardized to prove the viability of the cells producing anti-freeze proteins. 1) Two strains were tested TOP10 and JM109. Using strains transformed with AFP and untransformed (as control)
2) The strains were placed at -20 ° C for 24 hours
3) Thaw for 1 hour on ice
4) Inoculated in plates in a dilution of 10 ^ -5 (cycle 0)
5) The cells were frozen for 1 hour (-20 °C)
6) Afterwards they were placed 1 hour on ice and inoculated in a dilution of 10 ^ -7 (cycle 1)
7) The cells were frozen for 1 hour
8) Thaw for 1 hour on ice and inoculated in a dilution of 10 ^ -7 (cycle 2)
9) Freeze for 2 hour
10) Thaw for 1 hour on ice and inoculated in a dilution of 10 ^ -5 (cycle 3)
11) Freeze for 1 hour
12) Thaw for 30 minutes on ice inoculated in a dilution of 10 ^ -7 (Cycle 4)
13) Freeze for 1 hour
14) Thaw for 30 minutes on ice inoculated in a dilution of 10 ^ -7 (Cycle 5)
13) Incubate at 37 ° C
Results
The plates inoculated with transformed cells did not show growth while the non-transformed did show growth.
Experiment 3
The protocol was re-standardized one last time to prove the viability of the cells producing anti-freeze proteins.
1) Two strains were tested TOP10 and JM109. Using strains transformed with AFP and untransformed (as control), and also induced cells with arabinose+salt and arabinose by itself and non-induced cells to prove the effect of the inductor.
2) Cells were induced to 32°C for 12 hours; with arabinose+salt, arabinose, and non-induced cells.
3) The strains were placed at -20 ° C for 24 hours
4) Thaw for 3 hour on ice
5) Inoculated in plates in a dilution of 10 ^ -5 (cycle 0)
6) The cells were frozen for 4 hours (-20 °C)
7) Afterwards they were placed 3 hours on ice and inoculated in a dilution of 10 ^ -5 (cycle 1)
8) The cells were frozen for 2 hours
9) Thaw for 1 hour on ice and inoculated in a dilution of 10 ^ -3 (cycle 2)
10) Freeze for 1 hour
11) Thaw for 30 min on ice and inoculated in a dilution of 10 ^ -3 (cycle 3)
12) Incubate at 37 ° C
Results
Results were observed in all plates (See Results)
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