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Team:Tokyo Tech/Project/Apple Reporter
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[[Image:tokyotech_Fig. 1.1.9. zeaxanthin.jpg|right|200px|Fig. 1.1.9. zeaxanthin]] | [[Image:tokyotech_Fig. 1.1.9. zeaxanthin.jpg|right|200px|Fig. 1.1.9. zeaxanthin]] | ||
| - | [ | + | [[→more information about astaxanthin]] |
| - | Our team synthesized astaxanthin to make the pathway reach the end by assembling ''crtW'' and zeaxanthin synthesis constructs. (→[http://partsregistry.org/wiki/index.php?title=Part:BBa_K395706 BBa_K395706]) | + | 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> | ||
| + | [[Image:tokyotech_Fig. 2-1-10 1BBa_K395706.jpg|center|500px|thumb|S2]] | ||
| + | [[Image:tokyotech_Fig. 2-1-11 astaxanthin pellet and plate.jpg|left|310px|Fig. 2-1-11 astaxanthin pellet and plate]] | ||
| + | ~acetone extract and vacuum concentration~ [[→protocol]] | ||
| + | [[Image:tokyotech_Fig. 2-1-12 astaxanthin extract.jpg|right|310px|Fig. 2-1-12 astaxanthin extract]] | ||
| - | + | [[Image:tokyotech_table. 2.1.3.jpg|500px|center|thumb|table<2>]] | |
===Separation of carotenoids with TLC=== | ===Separation of carotenoids with TLC=== | ||
Revision as of 13:48, 27 October 2010
2-1 Color
Contents |
Abstract
Our team synthesized β-carotene, zeaxanthin and astaxanthin under several conditions (strain, construct,…etc) to confirm the functions. Particularly, astaxanthin is the final metabolite of the carotenoid synthetic pathway.
We synthesized β-carotene and zeaxanthin by assembling crtEBIY and crtZ of existing BioBrick parts. Though the part crtZ has been unconfirmed, we confirmed it for the first time in iGEM.
In addition, we succeeded in synthesizing astaxanthin by introducing the new BioBrick part crtW. Therefore, the carotenoid synthetic pathway reached the end.
After comparing the functions, we performed Thin Layer Chromatography (TLC) to confirm the products of β-carotene, zeaxanthin, astaxanthin were synthesized.
~carotenoid synthetic pathway~
Introduction
Carotenoids are natural organic pigments in plants and bacteria. Synthetic carotenoid pigments colored yellow, red or orange represent about 15-25% of the cost of production of commercial feed. Carotenoids are terpenoid based on a structure having the chemical formula C40H56. There are about 600 carotenoids, which perform a range of functions. For example, light energy absorption, protection against photo-damage, acting as antioxidants, and as precursor to other organic compounds such as vitamin A. However, astaxanthin is not converted to vitamin A in the human body. Too much vitamin A is toxic for a human, but astaxanthin has lower toxicity.
Previous year, our team has completed melanin synthetic pathway. Since the carotenoid synthetic pathway is huge, BioBrick Registry doesn’t cover all parts to complete the pathway. This year, we introduced a new BioBrick part to synthesize more various kinds of carotenoids. And we succeeded in synthesizing astaxanthin, one of the final metabolite of the pathway.
Furthermore, when the synthetic pathway is completed, it becomes able to change the color gradually by controlling the production of several carotenoids.
Results
β-carotene synthesis
→more information about β-carotene
Our team synthesized β-carotene under several conditions to confirm the activity of BioBrick parts designed by Cambredge 2009. (See more...)
zeaxanthin synthesis
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 . (→BBa_K395701, BBa_K395702, BBa_K395704)
We constructed two types of zeaxanthin synthetic construct.
One is that crtZ(BBa_I742158) and crtEBIY(BBa_K274210) were assembled on a single plasmid.(We call this construct “single plasmid construct”.) The other is that crtZ and crtEBIYwere assembled on different plasmids.(we call this type of constructs “double plasmids construct”.) We made 5 combinations shown in table<1>.
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.）
From the result, we decided to use pBad/araC promoter and double plasmids construct.
astaxanthin synthesis
→more information about astaxanthin
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. (→BBa_K395706), We also examine the production controlled by activity of pBad/araC promoter.
The activity of this promoter is measured in detail on another page. →click
We made 4 combinations shown in table<2>
~acetone extract and vacuum concentration~ →protocol
Separation of carotenoids with TLC
We performed thin layer chromatography (TLC) to confirm the products of β-carotene, zeaxanthin, astaxanthin were synthesized. (See more...)
Reference
