Team:Tokyo Tech/Project/Apple Reporter/Results

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==Results==
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==Protocols==
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===The work flow to TLC from culture===
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1. The cells were cultured over night.
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===&beta;-carotene synthesis===
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2. 50ul bacterial sution was taken from culture 1 and moved into 50ml LB culture with appropriate antibiotic.
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[[Image:tokyotech_Fig. 1.1.2.1. &beta;-carotene.jpg|right|200px|Fig. 1.1.2. &beta;-carotene]]
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Inducer was added,if necessary.
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[http://en.wikipedia.org/wiki/Beta-Carotene →more information about &beta;-carotene]
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3. The cells were incubated at 37℃24hour.
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Our team synthesized &beta;-carotene under several conditions to confirm the activity of BioBrick parts designed by Cambredge 2009.  
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We used ''crtEBIY'' (BBa_K274210) to synthesize &beta;-carotene. We compared the plasmids  containing pSB3K3 (low-copy vector) with the plasmids containing pSB1A2 (high-copy vector) from the production. We also introduced the low-copy plasmid into ''E. Coli''  strain MG1655, JM109 and DH5α respectively.
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4. After the O.D reach 2.0, culture was centrifuged for 10minutes at
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4℃, 6000rpm,10min
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We found that MG1655 and JM109 produce more &beta;-carotene than DH5α in liquid culture and that MG1655 grow better than JM109 on plate culture. These results indicated that MG1655 is the best of the three strains to treat. For this reason, we decided to use only MG1655 to extract pigment.
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5. Supernatant was removed by decanting. The remainder was added with 1 ml ddH<sub>2</sub>0 and vortexed.
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We also made special competent cell, MG1655 and JM109, having a plasmid ''crtEBIY''; pSB1A2 or ''crtEBIY''; pSB3K3) for zeaxanthin and astaxanthin synthesis.(→[https://2010.igem.org/Image:Tokyotech_Fig._1.1.7.1.BBa_K395704.jpg figure1], [https://2010.igem.org/Image:Tokyotech_Fig._2-1-10_1BBa_K395706.jpg figure2])
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6. Moved into 2ml tube by using P1000 pipetter at least twice.
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~acetone extract~
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[[→protocol]]
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[[Image:tokyotech_Fig. 1.1.3. &beta;-carotene extract.jpg|300px|left|thumb|Fig. 2-1-3. &beta;-carotene extract]]
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[[Image:tokyotech_Fig. 1.1.4. &beta;-carotene plate.jpg|300px|right|thumb|Fig. 2-1-4. &beta;-carotene plate]]
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[[Image:tokyotech_tokyotech_Fig. 1.1.5. note.jpg|500px|center|tokyotech_Fig. 1.1.5. note]]
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===zeaxanthin synthesis===
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[[Image:tokyotech_Fig. 1.1.6. zeaxanthin.jpg|right|200px|Fig. 1.1.6. zeaxanthin]]
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[[→more information about zeaxanthin]]
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Our team synthesized zeaxanthin by several kinds of construct to confirm the activity of BioBrick parts designed by Cambredge 2009 and Edinburgh 2007. By assembling the ''crtZ'', ''crtEBIY'' and appropriate promoters, we constructed new BioBrick parts to synthesize zeaxanthin and compared their function .
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(→[http://partsregistry.org/wiki/index.php?title=Part:BBa_K395701 BBa_K395701], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K395702 BBa_K395702], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K395704 BBa_K395704])
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We constructed two types of zeaxanthin synthetic construct.  
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One is that ''crtZ''(BBa_I742158) and ''crtEBIY''(BBa_K274210) were assembled on a single plasmid.(We call this construct “single plasmid construct”.)
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7. Solution is centrifuged for 20minutes at 4℃, 14000rpm
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The other is that ''crtZ'' and ''crtEBIY''were assembled on different plasmids.(we call this type of constructs “double plasmids construct”.)
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We made 5 combinations shown in table<1>.
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Constructs containing pBad/araC promoter on high-copy vector show the best pruduction and double plasmids construct shows as much pruduction as single plasmid construct under same promoter conbination. Construct with pBad/araC promoter shows better production than that with plac promoter. Construct that ''crtEBIY'' on high-copy vector and ''crtZ'' on low-copy vector shows as much expression as construct that ''crtEBIY'' on low-copy vector and ''crtZ'' on high-copy vector.（Actually, ③ showed a little more production than ⑤.）
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8. Supernatant was removed completely using pipetter. About 100ul of pellet is obtained.
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9. Pellet is added with 500ul acetone, and vortexed for 10 minutes to extract the pigment.
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10. Solution is centrifuged for 20minutes at 4℃, 14000rpm
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[[Image:tokyotech_Fig. 1.1.7.1.BBa_K395704.jpg|right|320px|thumb|S1]]
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11. Acetone solution was replaced into 2 ml tube.
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12. Acetone was removed using rotary evaporator.
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[[Image:tokyotech_Fig. 1.1.8.1. zeaxanthin pellet and plate.jpg|left|290px|Fig. 1.1.8.1. zeaxanthin pellet and plate]]
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13. 50 ul of Chloroform/Methanol solution(weight ratio of 9:1) was added.
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14. Sample was vortexed and spinned down.
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[[Image:tokyotech_table. 1.1.1.2.jpg|500px|center|thumb|table<1>]]
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15. The water layer was removed.
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16.  Spot the sample onto TLC silica gel plate, and developed by acetone/hexane solution.(weight ratio of 1:3)
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17. Spot was observed under visible light.
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From the result, we decided to use pBad/araC promoter and double plasmids construct.
 
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===astaxanthin synthesis===
 
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[[Image:tokyotech_Fig. 1.1.9. zeaxanthin.jpg|right|200px|Fig. 1.1.9. zeaxanthin]]
 
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[[→more information about astaxanthin]]
 
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Our team synthesized astaxanthin to make the pathway reach the end by assembling ''crtW'' and zeaxanthin synthesis constructs. After constructing new BioBrick parts to synthesize astaxanthin we compared their function. (→[http://partsregistry.org/wiki/index.php?title=Part:BBa_K395706 BBa_K395706]), We also examine the production controlled by activity of pBad/araC promoter.
 
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The activity of this promoter is measured in detail on another page. [[→click]]
 
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We made 4 combinations shown in table<2>
 
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[[Image:tokyotech_Fig. 2-1-10 1BBa_K395706.jpg|center|500px|thumb|S2]]
 
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[[Image:tokyotech_Fig. 2-1-11 astaxanthin pellet and plate.jpg|left|310px|Fig. 2-1-11 astaxanthin pellet and plate]]
 
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~acetone extract and vacuum concentration~ [[→protocol]]
 
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[[Image:tokyotech_Fig. 2-1-12 astaxanthin extract.jpg|right|310px|Fig. 2-1-12 astaxanthin extract]]
 
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[[Image:tokyotech_table. 2.1.3.jpg|500px|center|thumb|table<2>]]
 
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===Separation of carotenoids with TLC===
 
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We performed thin layer chromatography (TLC) to confirm the products of &beta;-carotene, zeaxanthin, astaxanthin were synthesized.
 
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The lanes are ①control(FPP), ②&beta;-carotene, ③zeaxanthin, ④astaxanthin from the left. We can tell ②,③ and ④ are yellow-orange, golden-yellow and red-orange pigment, respectively. These results are consistent with the Rf and color of &beta;-carotene, zeaxanthin, astaxanthin respectively and indicated that target products were synthesized as expected. [Reference: Carotenoids in crabs from marine and fresh waters of India ]
 
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[[Image:tokyotech_table. 2-1-3.jpg|400px|center|table<3>]]
 
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[[Image:tokyotech_Fig. 2-1-13 allTLC.jpg|500px|center|Fig. 2-1-13 allTLC]]
 
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&beta;-carotene and zeaxanthin
 
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[[Image:tokyotech_table. 2-1-4.jpg|315px|left|table<4>]]
 
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[[Image:tokyotech_Fig. 2-1-14 TLC &beta;-caroten zeaxanthin.jpg|315px|right|Fig. 2-1-14 TLC &beta;-caroten zeaxanthin]]
 
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astaxanthin
 
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[[Image:tokyotech_table 2-1-5-1.jpg|320px|left|table<5>]]
 
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[[Image:tokyotech_Fig. 2-1-15 TLC astaxanthin.jpg|300px|right|Fig. 2-1-15 TLC astaxanthin]]
 
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Not only astaxanthin but also another pigments were priduced at Ⅱ.
 
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We regarded them as intermediary metabolites between zeaxanthin and astaxanthin.
 
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==Protocol==
 
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===The work flow to TLC from culture===
 
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Latest revision as of 14:22, 27 October 2010

iGEM Tokyo Tech 2010 "E.coli with Humanity"

Protocols

The work flow to TLC from culture

1. The cells were cultured over night.

2. 50ul bacterial sution was taken from culture 1 and moved into 50ml LB culture with appropriate antibiotic. Inducer was added,if necessary.

3. The cells were incubated at 37℃24hour.

4. After the O.D reach 2.0, culture was centrifuged for 10minutes at 4℃, 6000rpm,10min

5. Supernatant was removed by decanting. The remainder was added with 1 ml ddH20 and vortexed.

6. Moved into 2ml tube by using P1000 pipetter at least twice.

7. Solution is centrifuged for 20minutes at 4℃, 14000rpm

8. Supernatant was removed completely using pipetter. About 100ul of pellet is obtained.

9. Pellet is added with 500ul acetone, and vortexed for 10 minutes to extract the pigment.

10. Solution is centrifuged for 20minutes at 4℃, 14000rpm

11. Acetone solution was replaced into 2 ml tube.

12. Acetone was removed using rotary evaporator.

13. 50 ul of Chloroform/Methanol solution(weight ratio of 9:1) was added.

14. Sample was vortexed and spinned down.

15. The water layer was removed.

16. Spot the sample onto TLC silica gel plate, and developed by acetone/hexane solution.(weight ratio of 1:3)

17. Spot was observed under visible light.


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