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Team:Tec-Monterrey/antifreeze/results
From 2012.igem.org
ANTIFREEZE
The antifreeze protein, transformed into BL21 Star and TOP10 F’, proved to express the protein when seen at the Tris-Tricine SDS-PAGE. Knowing that the RiAFP is present, what is next is to prove its activity. To prove the activity of the RiAFP, a massive 40-treatment experiment with 3 repetitions to consider variance was done and its respective analysis was made.
Using two different strains, BL21 star and TOP10F’, 4 different numbers of freeze-thaw cycles ranging from 0 to 3, absence and presence of inductors, 2 different inductor treatment (Arabinose+NaCl and Arabinose), and using transformed and non-transformed cells were all the different aspects we took into account when doing the experiment.
When the experiment finished, the results were processed doing linear regressions for all individual treatments. Also, most importantly, analysis of variance tests (ANOVA) were done to compare any significant difference between the treatments of interests.
In general, what we observe from the ANOVA tests was that in general, TOP10F’ resulted to be a better strain to resist cryopreservation than BL21 Star. Also, we noticed that Arabinose alone seemed to be a better inductor at the first times of freeze-thaw cycles, latter, the number of colonies counted when the inductor of arabinose+NaCl was used was greater than in just Arabinose. This could happen for different reasons; the combination of arabinose and NaCl could act in the solution increasing its colligative propierties, enduring the harsh temperatures and preventing the culture from freezing totally. Nevertheless, independent from which inductor was used and which strain was transformed, or even if it was inducted or no, we have enough information to affirm that our RiAFP is being expressed and with a desired activity.
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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