Team:Tec-Monterrey/antifreeze/results

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.