Team:Tokyo Tech/Antifreeze

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==Introduction of AFP==
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==Achievement==
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Why fish living in the Arctic Ocean don’t die even the temperature of the sea is below freezing point?
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*We achieved to introduce AFP derived from mealworm into ''E.coli'' strains Origami 2 and confirm the expression of AFP in the strain.  
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Most of the life cannot live in the climate which temperature is below the freezing point because water in their body freeze and these freezing damages harm their bodies. Everyone may have thought about this question once. Then, how the fish survive in such a severe climate?
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[[Image:Tokyo_Tech_AFP_result.png|200px|thumb|Fig 1. Expression of Anti-freeze protein]]
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The clue of this question is a protein discovered in 1969.  It is called Antifreeze Protein.
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It was firstly discovered in a fish living in the Antarctic Ocean, and until today, AFP is discovered from many other creatures and studied much more deeply.  In this page, we will introduce AFP’s characteristics.
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==The characteristics of AFP==
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==Introduction ~Why do we need Anti-freeze protein(AFP)~ ==
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AFP is defined that a substance binds to single ice crystal specifically and inhibit its growth.  When the temperature is below 0℃, water become 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 that it lowers the freezing point while leaving the melting point unchanged. These characteristics of AFP is called Thermal Hysteresis (TH), AFP is superior to other substances, for it can greatly lower the freezing point.  So we thought that the creature which enables to produce AFP (in their body) can lower the freezing point of their body fluid and is able to be less damaged so that it will survive.
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The surface temperature of Mars is below freezing point of water almost all day.
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Most organisms can not survive in such a severe environment even if we successed to send them to the Mars.
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However, fish in the Arctic Ocean survive even though the temperature of the sea is below freezing points. They could survive because they have 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.  By producing AFP, bacteria will become surviving in Mars.
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==Why we noticed AFP?==
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==Material and Method==
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The surface of the Mars is below freezing point almost all day.
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Most of the life will die if we simply send them to the present Mars.
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So we planed to transform AFP to E.coli body.  If it can produce AFP, coli will survive in low temperature and we will use less energy to primary investment.
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We focused on 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, giving it specific TH activity about 10-100 times greater than that of fish AFP, 300-500 times greater than other chemical compounds.  We expressed this AFP in E.coli and examined its activity (and how to shift the mortality rate when fungus bodies are in a repeated cycle of freeze thaw).
+
 +
===The characteristics of Anti-freeze protein(AFP)===
 +
Anti-freeze protein(AFP) is defined as a protein that binds to a single ice crystal specifically and inhibits  growth of the ice. 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(such as 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''.
 +
[[Image:Tokyo_Tech_AFP.gif|160px|thumb|Anti-freeze protein <br/> (PDB code 1ezg)]]
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 +
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===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). (All of these plasmids were designed and constructed by Nao Nakatani.)
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===Expression and Detection===
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We introduced the AFP plasmids into the ''E.coli'' Origami 2 strain (Novagen) and incubated 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  48 h at 15 ℃ after induction 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. Then, the gel was stained by CBB (Coomassie Brilliant Blue)(Fig 1).
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==Experiments==
 
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Designs of vectors
 
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We assembled the cording sequence of AFP synthetically from GENEART.  This gene digested with ClaⅠ, then it was integrated in the downstream of promoter, RBS, and eGFP for fusion protein.  In addition, we put a His tag for purification at the end of the insert.  This series of parts is on a Low Copy vector, pSB3K3.  We constructed several parts which differ in only promoter sequence.  We used constitutive PlacIq, constitutive in the presence of T7 RNA polymerase T7 promoter. Furthermore, inducible sequence LacO was added to T7 promoter.
 
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==Expression and Purification==
 
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We transformed the AFP vectors into the E.coli Origami2 strain (Novagen).  Single colonies were picked up from the freshly transformed cells and inoculated into LB medium containing 1.2μg/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 1h.  IPTG was added to a final concentration of 0.5mM to over-express the eGFP-AFP fusion protein.  Then cultures were shaken for a further 48h at 15℃ and harvested by centrifugation.  These fungus bodies were divided and some were checked for protein expression by SDS-PAGE and others were purified.  The x g of purified protein was dissolved in Yml of Milli-Q and checked its freezing point.
 
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==Measure of the freezing point==
 
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The purified AFP was dissolved into Milli-Q to Xmg/ml.  1ml of the aqueous solution was poured in a polymer test tube and layered 1ml of oil.  Thermometer was attached to the outside the tube then the device was put into a thermostat bath.  Temperature was slowly lowered (1℃/min) and we recorded that of inner solution.  The series of this operation was tried about 30 times.  We calculated the average of the freezing point, and defined as (E.coli produced) AFP’s freezing point.
 
==Results==
==Results==
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Expression of antifreeze protein
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===Expression of antifreeze protein===
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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 x kDa, and AFP-EGFP fusion protein is x kDa. The band of 3rd lane revealed around x kDa. So we regarded the protein as Antifreeze protein.
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The fusion proteins were (readily) detected by SDS–PAGE stained with CBB .  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 Anti-freeze protein.
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[[Image:Tokyo_Tech_AFP_result.png|200px|thumb|Fig 1.]]
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==Assessment the activity of antifreeze protein==
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We checked expression of AFP other way. We incubated ''E.coli'' Origami 2 which was introduced AFP 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).
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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.
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(This experiment done by Wakabayashi)
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[[Image:Tokyo_Tech_AFP_result.png|200px|thumb|Fig 1. Expression of Anti-freeze protein]]
==Discussion==
==Discussion==
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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 confirmed of the expression of the AFP in soluble fraction with 15 ℃ incubation. Therefore the expressed AFP is thought to function. Our next aim is to confirm whether the bacteria expressing AFP survive under low temperature like Mars environment. According to Yue and Zhang, addition of AFP into culture increased the survival rate of ''E.coli'' under low temperature. Therefore the expression of AFP might adapt bacteria to survive on the Mars environment.
 +
In our short term approach for terrforming of Mars, there are Heat Energy Injection term and Heat Energy Production term. So we supposed that energy injection from earth contribute to terrforming of Mars, but introducing of AFP will raise efficiency of terraforming of Mars more.
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==Reference==
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==References==
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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
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* 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
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C. Yue, Y. Zhang, Cloning and expression of Tenebrio molitor antifreeze protein in Escherichia coli, Molecular Biology Rep (2009) 36:529-536
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* C. Yue, Y. Zhang, Cloning and expression of ''Tenebrio molitor'' antifreeze protein in ''Escherichia coli'', Molecular Biology Rep (2009) 36:529-536

Latest revision as of 03:30, 22 October 2009

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Contents

Achievement

  • We achieved to introduce AFP derived from mealworm into E.coli strains Origami 2 and confirm the expression of AFP in the strain.
Fig 1. Expression of Anti-freeze protein

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 because they have 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. 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 as a protein that binds to a single ice crystal specifically and inhibits growth of the ice. 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(such as 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.

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). (All of these plasmids were designed and constructed by Nao Nakatani.)

Expression and Detection

We introduced the AFP plasmids into the E.coli Origami 2 strain (Novagen) and incubated 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 48 h at 15 ℃ after induction 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. Then, the gel was stained by CBB (Coomassie Brilliant Blue)(Fig 1).



Results

Expression of antifreeze protein

The fusion proteins were (readily) detected by SDS–PAGE stained with CBB . 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 Anti-freeze protein.

We checked expression of AFP other way. We incubated E.coli Origami 2 which was introduced AFP 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). (This experiment done by Wakabayashi)

Fig 1. Expression of Anti-freeze protein

Discussion

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 confirmed of the expression of the AFP in soluble fraction with 15 ℃ incubation. Therefore the expressed AFP is thought to function. Our next aim is to confirm whether the bacteria expressing AFP survive under low temperature like Mars environment. According to Yue and Zhang, addition of AFP into culture increased the survival rate of E.coli under low temperature. Therefore the expression of AFP might adapt bacteria to survive on the Mars environment. In our short term approach for terrforming of Mars, there are Heat Energy Injection term and Heat Energy Production term. So we supposed that energy injection from earth contribute to terrforming of Mars, but introducing of AFP will raise efficiency of terraforming of Mars more.

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
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