Team:Tokyo Tech/Antifreeze

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Safety

Achievement

We achieved to introduce AFP derived from mealworm into E.coli strains Origami 2 and confirm the expression of AFP in the strain. In addition, in vitro avtivity of AFP was confirmed.

Anti-freeze protein
PDB code 1ezg

Introduction ~Why do we need Anti-freeze protein(AFP)~

The surface temperature of Mars is below freezing point of water almost all day. Most organisms can not survive in such a severe environment even if we successed to send them to the Mars. However, fish in the Arctic Ocean survive even though the temperature of the sea is below freezing points. They could survive due to anti-freeze protein. Anti-freeze protein would enable organisms to surbvive in low temperature. Therefore, we tried to introduce Anti-freeze protein(AFP) into bacteria body. By producing AFP, bacteria will become surviving in Mars.

Material and Method

The characteristics of Anti-freeze protein(AFP)

Anti-freeze protein(AFP) is defined that a protein binds to single ice crystal specifically and inhibits its growth. When the temperature is below 0 ℃, water form ice nucleation. Then it grows up to bigger ice block. In the presence of AFP, on the other hand, AFP binds the ice nucleation very specifically and inhibits growing. For that reason AFP is thought to lower the freezing point while havinng no effect on the melting point. This characteristics of AFP is called Thermal Hysteresis (TH), AFP is superior to other substances(for example NaCl), for it can greatly lower the freezing point. Then we thought that the organisms with AFP can lower the freezing point of their body fluid and is able to be less damaged.

We decided to use the AFP of the yellow mealworm Teneblio molitor. This AFP consists of 84 amino acid, right handed β-helix with 12 residues per coil. This protein has many intramolecular disulfide bonds and a rigid array of threonine side chains, which give specific TH activity about 10-100 times greater than that of fish AFP, and 300-500 times greater than those of other chemical compounds. We expressed this AFP in E.coli and examined its activity (and how to shift the mortality rate when bacteria bodies are in a repeated cycle of freeze thaw).

Anti-freeze protein
(PDB code 1ezg)

Designs of plasmids

We obtained the coding sequence of AFP which was chemically synthesized from GENEART. This gene digested with ClaⅠ was inserted into the downstream of promoter, RBS, and EGFP. In this plasmid, AFP and EGFP formed a fusion protein. In addition, we put a His tag for purification at the end of the fusion protein. This series of parts is assembled on a Low Copy vector, pSB3K3. We constructed several parts which differ in only promoter sequence. We use placIq promoter(BBa_K193207) which is constitutive promoter, T7 promoter(BBa_K193208), and inducible sequence LacO was added to T7 promoter(BBa_K193209).

Expression and Detection

We introduced the AFP plasmids into the E.coli Origami 2 strain (Novagen) and incubatede 12 h at 37 ℃. Single colony was picked up from the transformed cells and inoculated into LB medium containing 30 μg/ml kanamycin. Cultures were grown at 37 ℃ with shaking (about 180rpm) to OD=0.5. The temperature was lowered to 15 ℃ and cultures were shaken for 1 h. After 1 h shaking, we added IPTG(0.5 mM final) to induce lacO. Then cultures were shaken for a further 48 h at 15 ℃ and harvested by centrifugation. After removing LB and adding PBS, culture boiled 5 minutes at 95 ℃ with 2×SB+2ME. SDS-PAGE electrophoresis at 120 V for 90 minutes(Fig 1).

Measure of the freezing point

The purified AFP was dissolved into Milli-Q to X mg/ml. We made solutions of five different concentration. 1ml of the aqueous solution was poured in a polymer test tube and layered 1ml of cilicon oil. Thermometer was attached to the outside the tube then the device was put into a thermostat bath. Temperature was slowly lowered (1.5 ℃/min) and we recorded the temperature of inner solution. The series of this operation was tried 3 times. We calculated the average of the freezing point, and defined the average as AFP’s freezing point.

Results

Expression of antifreeze protein

The fusion proteins were (readily) detected by SDS–PAGE stained with CBB (Coomassie Brilliant Blue). The result is shown Fig 1. 1st and 2nd lanes are EGFP, 3rd and 4th lane are AFP-EGFP fusion protein. 1st and 3rd lanes with IPTG induction, and 2nd and 4th lanes were without IPTG induction. Molecular weight of EGFP-His is about 30 kDa, and AFP-EGFP fusion protein is about 40 kDa. The band of 3rd lane revealed around 40 kDa. So we regarded the protein as Antifreeze protein.

Fig 1. Expression of Anti-freeze protein

Assessing the activity of antifreeze protein

We measured TH of a solution of anti-freeze protein purified from Origami 2. Calorimetric measurements of anti-freeze protein in Milli-Q water revealed that the protein depresses the freezing point. Supercooled water gave a sharp freezing endotherm with an onset of x °C, whereas water with TmTHP showed an endotherm with an onset of x °C. So the anti-freeze protein has an activity of TH.

Discussion

We checked expression of anti-freeze protein in other way. We incubated E.coli Origami 2 which was introduce anti-freeze protein plasmid for 24 h at 37 ℃. The result of SDS-PAGE electrophoresis, the band around 40 kDa was darker than the band of incubation of at 15 ℃(not shown). However, Checking solubilization of the each proteins, there weren’t any solubilizing protein at 37 ℃, but the protein at 15 ℃ were solubilized half amount of the protein which was expressed(not shown). Incubating E.coli introduced AFP at 37 ℃, anti-freeze protein would be expressed excessively, and E.coli would form inclusion bodies. Anti-freeze protein forming inclusion bodies wouldn’t have its activity and function to bind ice crystals.

In any case, we confirmed of the expression of the AFP. What should we do next is checking bacteria into which has been introduced AFP would survive in low temperature like Mars environment. AFP is the protein that fish living in the Arctic Ocean have and organisms which have been living on the extremely low temperature must need AFP to survive. According to C. Yue, Y. Zhang (Molecular Biology Rep (2009)), Comparing culture with AFP to culture without AFP, after lowering it, the number of surviving E.coli would have been different between two cultures. So if there were confirmation of surviving bacteria in low temperature by introducing AFP, it would be useful to terraforming of Mars. Conclusion, terrforming of Mars, there must’ve need artificial energy to warm environment and organisms, introducing of AFP would enable it to less energy

References

M. Bar, R. Bar-Ziv, T. Scherf, D. Fass, Efficient production of a folded and functional, highly disulfide-bonded β-helix antifreeze protein in bacteria, Protein Expression & Purification, 2006
C. Yue, Y. Zhang, Cloning and expression of Tenebrio molitor antifreeze protein in Escherichia coli, Molecular Biology Rep (2009) 36:529-536

@@ Line 38: / Line 38: @@
 ==Discussion==
-We checked expression of anti-freeze protein other protocol. We incubated ''E.coli'' Origami 2 which was introduce anti-freeze protein plasmid for 24 h at 37 ℃. The result of SDS-PAGE electrophoresis, the band around 40 kDa was darker than the band of incubation of at 15 ℃(not shown). However, Checking solubilization of the each proteins, there weren’t any solubilizing protein at 37 ℃, but the protein at 15 ℃ were solubilized half amount of the protein which was expressed(not shown). Incubating ''E.coli'' introduced AFP at 37 ℃, anti-freeze protein would be expressed excessively, and ''E.coli'' would form inclusion bodies. Anti-freeze protein forming inclusion bodies wouldn’t have its activity and function to bind ice crystals.
+We checked expression of anti-freeze protein in other way. We incubated ''E.coli'' Origami 2 which was introduce anti-freeze protein plasmid for 24 h at 37 ℃. The result of SDS-PAGE electrophoresis, the band around 40 kDa was darker than the band of incubation of at 15 ℃(not shown). However, Checking solubilization of the each proteins, there weren’t any solubilizing protein at 37 ℃, but the protein at 15 ℃ were solubilized half amount of the protein which was expressed(not shown). Incubating ''E.coli'' introduced AFP at 37 ℃, anti-freeze protein would be expressed excessively, and ''E.coli'' would form inclusion bodies. Anti-freeze protein forming inclusion bodies wouldn’t have its activity and function to bind ice crystals.
 In any case, we confirmed of the expression of the AFP. What should we do next is checking bacteria into which has been introduced AFP would survive in low temperature like Mars environment. AFP is the protein that fish living in the Arctic Ocean have and organisms which have been living on the extremely low temperature must need AFP to survive. According to C. Yue, Y. Zhang (Molecular Biology Rep (2009)), Comparing culture with AFP to culture without AFP, after lowering it, the number of surviving ''E.coli'' would have been different between two cultures. So if there were confirmation of surviving bacteria in low temperature by introducing AFP, it would be useful to terraforming of Mars. Conclusion, terrforming of Mars, there must’ve need artificial energy to warm environment and organisms, introducing of AFP would enable it to less energy