Difference between revisions of "Team:Nagahama/Design"

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{{Nagahama}}
 
{{Nagahama}}
  
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=Prototype=
  
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Our project is Flavorator. We show its system working  under real-world conditions  in India . Because India has a problem about storage of foods.  We want to spread Flavorator  to the world , and we thought that our Flavorator can solve such as its problem. This is why we choose India.
  
= '''Protocols''' =
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According to Assocham that is Joint Chamber in India ,
Our Lab's Protocols
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30 percent of the total harvest of agricultural crops in India have been discarded by lack of refrigerated storage facility .
 +
Refrigerated storage facility is insufficient in the retail market in rural areas and suburbs.In 2011, Refrigerator penetration  in the cities is 43.8% , and the villages is 9.4%(fig1). The penetration  in rural areas is much smaller than the city . Its cause is considered that  supply of electricity are not yet ready. To solve this problem, there is a need for method of preserving  food products that do not require electricity in rural and suburban markets. Our Flavorator does not require electricity. So, we considered that it is used in situations such as the India, and designed Flavorator.
  
== Medium ==
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[[File:India graph.png|450px|thumb|none|fig1:Refrigerator Penetration rate in Inindia ]]
  
=== LB medium (100 mL liquid) ===
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==Design==
  
1.Measure 1g Tripton
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This is the design of Flavorator (fig2)
  
2.Measure 0.5g Yeast Extract
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・Container of medium is attached in above the box , fragrances is produced from there (①). As a result, fragrance circulates without electricity (③). This phenomenon is called natural convection.
  
3.Measure 1g Nacl
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・It must to keep temperature in Flavorator for recombinants  activity. A layer of vacuum  suppress the change of temperature in Flavorator because it work as a heat insulator.(②)
  
4.Add 100mL H2O
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・The container of the medium is covered by a triple filter, it prevents the contact of recombinants and foods (④). Recombinants are larger than the mesh of filter, so they can't go through a triple filter , but fragrance is able to pass through  because its molecules are smaller than the mesh (fig3).
  
5.autoclave(121℃ 20min)
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・Fragrance can be chosen by changing recombinants to others.
  
=== 2×YT medium (100mL liquid)===
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[[File:Fravorator_prototype.png|450px|thumb|none|fig2:Design of Fravorator ]]
  
1.Measure 1.6g Tripton
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[[File:Filter修正.png|400px|thumb|none|fig3:filer]]
  
2.Measure 1g Yeast Extract
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==Is it possible to preserve foods by Flavorator?==
  
3.Measure 0.5g Nacl
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We experimented  preserving food by fragrance with antimicrobial activity. fig4 is preservation of pork by garlic, and fig5 is preservation of bread by geraniol. The mashed garlic and geraniol releases fragrant substances that has antibacterial activity.  
  
4.Add 100mL H2O
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From these result, we considered that foods can be preserved in Flavorator if our recombinants release a large amount of fragrance.  
  
5.autoclave(121℃ 20min)
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[[File:にんにく.jpg|300px|thumb|left|Fig.4:Storage of pork by garlic.
  
 
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(A) was put mashed garlic 5g in the bottom of box. Garlic does not touch pork. We left the two pork in the box at 18℃ for 2 months.2 months later, Left pork (A) didn’t change the color. Right pork (B) changed Pink to Brown.]][[File:パン.jpg|サイズpx|thumb|center|Fig.5:Storage of bread by geraniol.
== DNA work ==
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A:ddH2O(300 μl)B:geraniol(stock solution, 300 μl)
 
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=== Agarose gel(100mL) ===
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Method of Making 0.7% Agarose gel
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1.Measure 0.7g Agarose
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2.Add 100mL TAE buffer
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3.Heat(till agarose melted)*We used a microwave oven.
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4.Pur agarose into a gel maker
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5.Set a comb
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6.Wait till agarose curdles
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7.Pull an comb
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=== Genome DNA extraction ===
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↓Cultivate ''E. coli DH5α'' using LB medium 2ml×2 tubes O/N
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↓Centrifuge culture 1.5ml  (13,000rpm 4℃ 1min)
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↓Remove the culture
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↓+TNE buffer 1.0ml  (10mM Tris pH 8.0, 10 mM NaCl, 10 mM EDTA)
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↓vortex
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↓Centrifuge cell suspension  (13,000rpm 4℃ 2min)
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↓Remove  supernatant
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↓+TNE buffer including 1% Triton X-100  270µl
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↓Suspend cell gently
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↓+5mg/ml Lysozyme solution 30µl (0.15g Lyzozyme + 30µl sterile water)
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↓Reaction 37℃ 30min
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↓+ 20mg/ml Proteinase K (fin.con: 1mg/ml)    TaKaRa Code:9033
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↓Reaction 65℃ 2h
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↓+Phenol chloroform 300µl ...(1)
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↓Mix the solution gently ...(2)
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↓Centrifuge (13,000rpm 4℃ 8min) ...(3)
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↓Transfer only water layer to new 1.5ml tube ...(4)
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↓Repeat (1)~(4) 2 times
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↓+3M Sodium acetate 30µl
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↓Mix gently
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↓+99.5% EtOH 750µl
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↓Mix gently
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↓Centrifuge (13,000rpm 4℃ 10min)
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↓Remove supernatant
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↓+70% EtOH 500µl
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↓Centrifuge (13,000rpm 4℃ 1min)
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↓Remove supernatant
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↓Dry up the pellet covering with aluminum foil at room temperature 30min
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↓+TE buffer 50µl
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↓Suspend DNA gently
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[[File:GenomeDNA_DH5a2.png]]
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=== Plasmids extraction ===
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=== PCR ===
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=== Transformation by heat shock===
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==Proof of concept(POC)==
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Result [https://2015.igem.org/Team:Nagahama/Experiments#Fragrance_of_wasabi here]
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==='''Fragrance of Garlic'''===
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We prepared 20g pork and 5g [https://en.wikipedia.org/wiki/Garlic garlic]. We left the pork for an hour on the table and grated garlics. We spread the garlic grated on the pork (A).  We left the two pork in the box at 18℃ for 2 month. Left pork (A) didn’t change the color. Right pork (B) changed Pink to Brown. We found from this result that fragrance of plants have antibacteril volatiles.
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==='''Fragrance of Wasabi'''===
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We prepared some rice cake and 10g [https://en.wikipedia.org/wiki/Wasabi wasabi]. We left the rice cake for an hour on the table and grated wasabi. We spread the wasabi grated on the rice cake (A). We left each rice cake in the box at 18℃ for 2 month. Rice cake of the above (A) didn’t change the color. Rice cake under (B) changed white to Black. We found from this result that fragrance of plants have antibacteril volatiles.
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==='''Fragrance of Geraniol'''===
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パン
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Storage of food by geraniol
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A:ddH2O(300 μl)
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B:geraniol(stock solution, 300 μl)
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Incubation: 35 days
 
Incubation: 35 days
 
Temperature: room temperature
 
Temperature: room temperature
Chopsticks dipped in suspension of ''Penicillium'' was put in the center of the bread.
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Chopsticks dipped in suspension of mold was put in the center of the bread.
 
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''Penicillium'' was cultured among the left box(A), and ''Penicillium'' wasn’t cultured among the right box(B), suggesting that ''Penicillium'' can’t be cultured in a state where geraniol is filled.
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This experiment indicate that geraniol might be the effect of suppressing the growth of bacteria
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== isoprenoid production ==
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Because of its high hydrophobicity and low volatility, decane was chosen to extract and solubilize farnesolFOH from the culture broth. The decane was overlaidy in the two-phase culture media, but it did not affect the cell growth., and FOH farnesol could be well solubilized in the decane phase with negligible volatile loss. We adopt used 1 mL of decane to overlayid to the 5 mL of culture broth. Two-phase The culture of E. coli JM109 (BBa_K165025) was carried out in 2YT medium containing 1% glycerol at 29°C for 48 h. The decane phase of the two-phase culture was collected to analyze the FOH content by GC-MS.
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==Analysis of ubiquinone-8 synthesized by ''E. coli''==
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1. Put ''E. coli'' cells in: methanol (7: 2).
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<br>
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2. Sonicate cells  for 30 sec. and cool down 30 sec. and collect supernatants. ( 6 times).
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<br>
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3. Spin down the supernatants at 12,000xg, 10min at 4 ℃, and  dry up acetone and methanol.
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<br>
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4. Add 400μl of chloroform : methanol ( 1 : 1 ) to the dried supernatants. Add equal volume 0.7% NaCl and spin down 12,000xg 5min at 4 ℃.
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<br>
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5. Extract the lower layer
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<br>
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6. Add 100μl of chloroform  : methanol (2: 1).
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<br>
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7. Spot the extracts on TLC plate and develop extracts  using benzene : acetone (7 93).
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== geraniol tolerant assay ==
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== GC ==
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== GC-MS ==
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Mold were cultured among the left box(A), and mold weren’t cultured among the right box(B).]]

Revision as of 04:42, 17 September 2015

Team Nagahama banner.jpg

Prototype

Our project is Flavorator. We show its system working under real-world conditions in India . Because India has a problem about storage of foods. We want to spread Flavorator to the world , and we thought that our Flavorator can solve such as its problem. This is why we choose India.

According to Assocham that is Joint Chamber in India , 30 percent of the total harvest of agricultural crops in India have been discarded by lack of refrigerated storage facility . Refrigerated storage facility is insufficient in the retail market in rural areas and suburbs.In 2011, Refrigerator penetration in the cities is 43.8% , and the villages is 9.4%(fig1). The penetration in rural areas is much smaller than the city . Its cause is considered that supply of electricity are not yet ready. To solve this problem, there is a need for method of preserving food products that do not require electricity in rural and suburban markets. Our Flavorator does not require electricity. So, we considered that it is used in situations such as the India, and designed Flavorator.

fig1:Refrigerator Penetration rate in Inindia

Design

This is the design of Flavorator (fig2)

・Container of medium is attached in above the box , fragrances is produced from there (①). As a result, fragrance circulates without electricity (③). This phenomenon is called natural convection.

・It must to keep temperature in Flavorator for recombinants activity. A layer of vacuum suppress the change of temperature in Flavorator because it work as a heat insulator.(②)

・The container of the medium is covered by a triple filter, it prevents the contact of recombinants and foods (④). Recombinants are larger than the mesh of filter, so they can't go through a triple filter , but fragrance is able to pass through because its molecules are smaller than the mesh (fig3).

・Fragrance can be chosen by changing recombinants to others.

fig2:Design of Fravorator
fig3:filer

Is it possible to preserve foods by Flavorator?

We experimented preserving food by fragrance with antimicrobial activity. fig4 is preservation of pork by garlic, and fig5 is preservation of bread by geraniol. The mashed garlic and geraniol releases fragrant substances that has antibacterial activity.

From these result, we considered that foods can be preserved in Flavorator if our recombinants release a large amount of fragrance.

Fig.4:Storage of pork by garlic. (A) was put mashed garlic 5g in the bottom of box. Garlic does not touch pork. We left the two pork in the box at 18℃ for 2 months.2 months later, Left pork (A) didn’t change the color. Right pork (B) changed Pink to Brown.
Fig.5:Storage of bread by geraniol. A:ddH2O(300 μl)B:geraniol(stock solution, 300 μl) Incubation: 35 days Temperature: room temperature Chopsticks dipped in suspension of mold was put in the center of the bread. Mold were cultured among the left box(A), and mold weren’t cultured among the right box(B).