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Team:Tec-Monterrey/antifreeze/results
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<p>Using a 96-well fluorescence plate (a black one) we filled some wells with supernatant from our induced <i>Pichia pastoris</i> with scFv IgE+GFP, some with the intracellular extract from the same culture, and the same treatments but for our <i>P.pastoris</i> with scFv His. The fluorescence plate for the experiment was designed as follows:</p> | <p>Using a 96-well fluorescence plate (a black one) we filled some wells with supernatant from our induced <i>Pichia pastoris</i> with scFv IgE+GFP, some with the intracellular extract from the same culture, and the same treatments but for our <i>P.pastoris</i> with scFv His. The fluorescence plate for the experiment was designed as follows:</p> | ||
| - | <br> < | + | <br> <img src ="https://static.igem.org/mediawiki/2012/c/ca/TECMTY_01.jpg"/> |
<p>Where GFP III, IV, V, VI are different <i>P.pastoris </i>strains expressing scFvIgE+GFP, Api is a <i>P.pastoris</i> strain expressing Api m 6, His is a strain from the same organism expressing scFv His, SN means supernatant , ICs refers to the soluble fraction of the intracellular extraction and Blank is just media without any strain. </p> | <p>Where GFP III, IV, V, VI are different <i>P.pastoris </i>strains expressing scFvIgE+GFP, Api is a <i>P.pastoris</i> strain expressing Api m 6, His is a strain from the same organism expressing scFv His, SN means supernatant , ICs refers to the soluble fraction of the intracellular extraction and Blank is just media without any strain. </p> | ||
Revision as of 17:45, 12 October 2012
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:
Where GFP III, IV, V, VI are different P.pastoris strains expressing scFvIgE+GFP, Api is a P.pastoris strain expressing Api m 6, His is a strain from the same organism expressing scFv His, SN means supernatant , ICs refers to the soluble fraction of the intracellular extraction and Blank is just media without any strain.
The results were organized on the following tables, the first one comparing the results between the different GFP strains and their fluorescence in supernatant and intracellular-soluble fractions and the second one comparing one of the GFP strains to the other ones.
In average, the results seem to mean that we indeed have a fluorescent protein on the desired strains and that it does exists more in the supernatant, thanks to the signal peptide is was designed to have. But just that is not enough to approve anything, to interpret these results with more confidence we need to do further mathematical analysis.
The ANOVA test was chosen because we have al the conditions needed for its application. So two ANOVA tests were done, the first one to see if there was any difference in fluorescence between the GFP strains and/or between the fraction (supernatant/ intracellular soluble), and the second to see the same difference but between one of the GFP strains and the His/Api extractions.
Thanks to the ANOVA 1 test, now we can conclude information with certainty. The ANOVA test table above shows that our statistic Fp for the source A is greater than the ft, this means that data from the source A (fraction) is significantly different with a 95% percent if confidence. The Fp of source B being not greater than its ft, means that there is no significant difference between the fluorescence of the GFP from different strains. Finally, the AB source having a Fp less than its ft means that there is no difference in combinations of source A and B, which is not of our interest. This means, that yes, we can conclude that we have more fluorescence in the supernatant and that all of our GFP strains are expressing the same with at least 95% of confidence.
The ANOVA 2 test was designed so that it would tell us if we had any difference in fluorescence between a GFP strain and the other non-GFP strains. Using the same logic that in ANOVA 1 test and comparing the Fp’s from the ft’s, we can conclude with at least 95% of confidence that our GFP is different in fluorescence that our other strains and again we can conclude that this protein is being secreted. In short, the GFP part of our scFv IgE+GFP does work and it is being secreted successfully!
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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