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Team:Tec-Monterrey/antifreeze/results
From 2012.igem.org
ANTIFREEZE
Production of RiAFP E.coli
E.coli Protocol
Tricine SDS-PAGE (%16) under reducing conditions.
Lane 1: E.coli (JM 109, BL21 STAR, Rosetta Gami) soluble fraction lysate post-IMAC 16.6 KDa Der f 2 (SES)
Lane 2 and Lane 3: Molecular weight marker.
Lane 4: E.coli (TOP 10, BL21 STAR) soluble fraction lysate post-IMAC 13.7 KDa RiAFP
IMAC purification of two different proteins (RiAFP & Der f 2) revelead that several metal affinity proteins prevent a clear visualization of our proteins of interest in SDS-PAGE. Moreover, RiAFP (13.7 KDa) was absen in the gel. We believe that in both proteins, their Histidine tags remained enclosed within the inner structure of the protein, restraining the purification of the proteins by affinity cromatography.
Viability assay of TOP10 F and BL21 STAR strains induced with two different promoters
In this experiment, BL21 STAR and TOP F' strains were transformed with RiAFP and treated with Arabinose +NaCl and Arabinose as inductors, measuring viability after each freeza-thaw cycle. Our most significant treatment was RiAFP +BL21 STAR strain induced with Arabinose +NaCl. We found out that the effect of RiAFP becomes more evident over the number of cycles.
ANOVA test to prove the significant effects of individual treatments on cell viability for each strain. Circle, cross and dash indicate possitive, negative and neutral effect respectively.
Increase of cell viability ratio between BL21 STAR transformed with RiAFP and BL21 STAR not transformed cells, over freezing-thawing cycles.
Conclusions RiAFP
RiAFP presence was not conclusively determined by SDS-PAGE; nevertheless, RiAFP viability tests demonstrated significant difference between positive and negative transformants, suggesting expression of the protein in the cells. Further experimentation with variable inductor concentrations would be needed to optimize cryopreservation conditions of RiAFP, as well as longer storage periods. Given the advantages of the RiAFP transformants, we speculate, increase in the competency of RiAFP (+) cells will be measured by comparing the transformation efficiency between positive and negative RiAFP-transformants.
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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