Team:Tokyo Tech/Project/Apple Reporter

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|<h2>'''Apple Reporter'''</h2>]]
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|[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System|Artificial Cooperation System]]
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|[[Team:Tokyo_Tech/Project/wolf_coliwolf|coli]]
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<table id="table-01">
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<td>[[Team:Tokyo_Tech|1 Graphic abstract]]<br>
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<th>2 Apple reporter<br>
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:2-1 Color  -YOU ARE HERE!-
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:[[Team:Tokyo_Tech/Project/Apple_Reporter2|2-2 Fragrance]]
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</th>
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<tr>
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<td>[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System|3 Artificial Cooperation System]]<br>
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:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|3-1 ''lux'' activation/repression promoter]]
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:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assay|3-2 resistance gene activation device]]
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:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|3-3 ''lux''I Assay]]
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:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modeling|3-4 modeling]]
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</td>
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</tr>
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<tr>
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<td>[[Team:Tokyo_Tech/Project/wolf_coli|4 wolfcoli overview]]<br>
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:[[Team:Tokyo_Tech/Project/wolf_coli/New_Series_of_PompC|4-1 New series of P''ompC'']]
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:[[Team:Tokyo_Tech/Project/wolf_coli/lacIM1|4-2 lacIM1 for band-detect network ]]
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:[[Team:Tokyo_Tech/Project/wolf_coli/System|4-3 Wolf coli system]]
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</td>
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</tr>
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</table>
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</center>
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<font size="5"><b>2-1 Color</b></font>
<!--　ここから始まるよ-->   
<!--　ここから始まるよ-->   
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==Color==
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==Abstract==
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=overview=
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Our team synthesized &beta;-carotene, zeaxanthin and astaxanthin under several conditions (strain, plasmid copy number,…etc) to confirm the functions of BioBrick parts designed by previous iGEM teams. Particularly, '''astaxanthin is the final metabolite of the carotenoid synthetic pathway.'''
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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 vitaminA.
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Production of pigment has been worked in iGEM, and quite a few parts for synthesis of carotenoid-related compounds already exist in BioBricks Registry. Since the carotenoid synthetic pathway is huge, BioBricks Registry doesn’t cover all parts to complete the pathway. This time, we intorduced some new BioBrick parts to synthesize more various kinds of carotenoids. And we succeeded in synthesizing astaxanthin, one of the final metabolite of the pathway. Also, astaxanthin is not converted to vitaminA in the human body. Too much vitamin A is toxic for a human, but astaxanthin has lower toxicity.
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Furthermore, as the synthetic pathway is completed, it becomes possible to change the hue gradually by controlling the production of several carotenoids.  
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We synthesized &beta;-carotene and zeaxanthin by assembling ''crtEBIY'' and ''crtZ'' of existing BioBrick parts. '''Though the part ''crtZ'' has been unconfirmed to produce zeaxanthin, we confirmed it for the first time in iGEM.'''
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~carotenoid synthetic pathway~
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In addition, we succeeded in synthesizing astaxanthin by introducing '''the new BioBrick part  ''crtW'''''. Therefore, the carotenoid synthetic pathway reached the end.
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[[Image:tokyotech_Fig. 1.1.1. carotenoid synthetic pathway.jpg|500px|Fig. 1.1.1. carotenoid synthetic pathway]]
 
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([[right|→our BioBrick parts]])
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[[Image:tokyotech_Fig. 1.1.1. carotenoid synthetic pathway.jpg|390px|left|thumb|Fig. 2-1-1. carotenoid synthetic pathway]]
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=works=
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[[Image:TLC work3.jpeg|right|210px|thumb|Fig.2-1-2.TLC  worked by Yumiko Kinoshita]]
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[[Image:tokyotech_Fig. 1.1.2.1. β-carotene.jpg|right|200px|Fig. 1.1.2. β-carotene]]
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・ β-carotene synthesis
 
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[[→more information about β-carotene]]
 
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Our team synthesized β-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 β-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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[https://2010.igem.org/Team:Tokyo_Tech/Parts &rarr;our BioBrick parts]
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[http://partsregistry.org/Part:BBa_K395704 &rarr;BBa_395704]
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[http://partsregistry.org/Part:BBa_K395706 &rarr;BBa_395706]
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==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 C<sub>40</sub>H<sub>56</sub>. There are about 600 carotenoids, which perform a wide range of functions. (For example, acting as antioxidants[https://2010.igem.org/Team:Tokyo_Tech/Project/Apple_Reporter#Reference], and as precursors to other organic compounds.) '''Among carotenoids, astaxanthin is not known to be converted to Vitamin A, which is converted from most of carotenoids and is toxic when its concentration too high.'''
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~acetone extract~
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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.'''
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[[→protocol]]
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[[Image:tokyotech_Fig. 1.1.3. β-carotene extract.jpg|300px|left|thumb|Fig. 1.1.3. β-carotene extract]]
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[[Image:tokyotech_tokyotech_Fig. 1.1.5. note.jpg|500px|center|thumb|Fig. 2-1-3. note]]
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==Results==
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[[Image:tokyotech_Fig. 1.1.4. β-carotene plate.jpg|300px|right|thumb|Fig. 1.1.4. β-carotene plate]]
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===&beta;-carotene synthesis===
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[[Image:tokyotech_Fig. 1.1.2.1. &beta;-carotene.jpg|right|200px|thumb|Fig.2-1-4.&beta;-carotene]]
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 +
Our team synthesized &beta;-carotene under several conditions to confirm the functions of BioBrick parts designed by Cambridge 2009. (we call it "crtEBIY plasmids".) ([https://2010.igem.org/Team:Tokyo_Tech/Project/Apple_Reporter#appendix See more...])
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===zeaxanthin synthesis===
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[[Image:tokyotech_Fig. 1.1.6. zeaxanthin.jpg|right|200px|thumb|Fig.2-1-5zeaxanthin]]
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Our team tried to synthesize zeaxanthin by using the constructed ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K395704 BBa_K395704])part to confirm the activity of BioBrick parts designed by Cambredge 2009 and Edinburgh 2007. We also confirmed zeaxanthin by TLC (Fig.2-1-6 spot III).
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By introduction of ''crtZ'' gene to the ''crtEBIY'' plasmid (we call this construct “single plasmid construct”, "''crtZ-crtEBIY''".), new spot appered on TLC plate (Fig.2-1-6 spot III). The spot shows lower Rf value than that derived from ''crtEBIY'' (Fig.2-1-6 spot II).  The Rf values of spots derived from ''crtEBIY'' and ''crtZ-crtEBIY'' corresponded to reported values of &beta;-caroten and Zeaxanthin, respectively.[https://2010.igem.org/Team:Tokyo_Tech/Project/Apple_Reporter#Reference] Thus we could characterize zeaxanthin production activity of crtZ. In addition, caracterization of the part ''crtZ'' is the first work in iGEM.
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In order to maximize zeaxanthin production, we designed some combinations of construct. We thought of another type of zeaxanthin synthetic construct where ''“crtZ”'' (BBa_I742158) and ''“crtEBIY”'' (BBa_K274210) were separated on different plasmids. (we call this type of constructs “double plasmids construct.”)
 +
After all, We made five combinations shown in table<1>, including [http://partsregistry.org/wiki/index.php?title=Part:BBa_K395701 BBa_K395701], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K395702 BBa_K395702] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K395704 BBa_K395704].
 +
Constructs containing pBad/araC promoter on high-copy vector show the best production and double plasmids construct shows as much production as single plasmid construct under same promoter combination. 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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For TLC, We selected the best three samples including the sample derived from single plasmid construct .
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In the consequence of TLC, we could confirm that these three samples were zeaxanthin.
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[[Image:S1-zeaxanthin.jpg|left|300px|thumb|S1 : zeaxanthin expression components]]
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[[Image:TLC work3.jpeg|right|300px|thumb|Fig.2-1-6 zeaxanthin TLC  worked by Yumiko Kinoshita]]
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[[Image:Tokyotech_zeaxanthin_synthesis.jpg|center|500px|thumb|Fig.2-1-7 table<1>　 zeaxanthin  worked by Yumiko Kinoshita]]
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===astaxanthin synthesis===
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[[Image:tokyotech_Fig. 1.1.9. zeaxanthin.jpg|right|200px|thumb|Fig.2-1-8 astaxanthin]]
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Our team synthesized astaxanthin for the first time in iGEM. Thus the carotenoid synthetic pathway reached the end. This work was done by assembling the new BioBrick part ''crtW'' and zeaxanthin synthesis constructs we designed. From the result of “zeaxanthin synthesis”, we decided to use pBad/araC promoter and double plasmids construct.
 +
After constructing the new BioBrick device ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K395706 BBa_K395706]) to synthesize astaxanthin, we characterize the function under the condition I in table<2>.  Thus we confirmed astaxanthin by TLC (Fig2-1-6 spot IV).
 +
By introduction of ''crtW'' gene to the ''crtZ'' plasmid (the second construct of five combinations in “zeaxanthin synthesis”,having pBad/araC promoter), new spot ((Fig2-1-6 spot IV) appeared on TLC plate. The spot shows lower Rf value than that derived from ''crtEBIY'' (II) and a little higher Rf value than that derived from ''crtZ-crtW''(III). All the Rf values of  spots corresponded to reported values  respectively.[https://2010.igem.org/Team:Tokyo_Tech/Project/Apple_Reporter#Reference [2]] Thus we succeeded in characterizing astaxanthin production activity of ''crtW''. ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K395705 BBa_K395705], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K395706 BBa_K395706])
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For the same reason as “zeaxanthin synthesis”, we examined under several conditions &rarr;table<2>.
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In the consequence of TLC, we could confirm that these four samples were astaxanthin.
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Not only astaxanthin but also another spots were produced at II.
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We regarded them as intermediary metabolites between zeaxanthin and astaxanthin.
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We found that MG1655 and JM109 produce more β-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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The activity of pBad/araC promoter is measured in detail on another page. [https://2010.igem.org/Team:Tokyo_Tech/Project/wolf_coli/lacIM1 &rarr;click]
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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.([[→figure]])
 
 +
[[Image:S2-astaxanthin.jpg|left|300px|thumb|S2 : astaxanthin expression component]]
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[[Image:tokyotech_tokyotech_Fig. 1.1.5. note.jpg|600px|center|tokyotech_Fig. 1.1.5. note]]
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[[Image:TLC work3.jpeg|right|300px|thumb|Fig.2-1-6 TLC astaxanthin  worked by Yumiko Kinoshita]]
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[[Image:Tokyotech_astaxanthin_synthesis.jpg|center|500px|thumb|Fig.2-1-9 astaxanthin  worked by Yumiko Kinoshita]]
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[[Image:tokyotech_Fig. 1.1.6. zeaxanthin.jpg|right|200px|Fig. 1.1.6. zeaxanthin]]
 
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・ zeaxanthin synthesis
 
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[[→more information about zeaxanthin]]
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acetone extract and vacuum concentration [https://2010.igem.org/Team:Tokyo_Tech/Project/Apple_Reporter#Materials_and_Methods &rarr;protocol]
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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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==Reference==
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(→[[BBa_K395701]], [[BBa_K395702]], [[BBa_K395704]])
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[1] Yousry M. Antioxidant Activities of Astaxanthin and Related Carotenoids. J. Agric. Food Chem. 2000, 48, 1150-1154
 +
[2]N.M. Sachindra, et al. Carotenoids in crabs from marine and fresh waters of India. LWT 38 (2005) 221–225
 +
[3]MISAWA N, et al. Structure and Functional Analysis of a Marine Bacterial Carotenoid Biosynthesis Gene Cluster and Astaxanthin Biosynthetic Pathway Proposed at the Gene Level. JOURNAL OF BACTERIOLOGY, Nov. 1995, p. 6575–6584 Vol. 177, No. 22
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We constructed two types of zeaxanthin synthetic construct.
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[4]Seon-Kang C, et al. Characterization of bacterial β-carotene Ketolases, CrtW,
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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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from Mrine Bacteria by Complementation Analysis in ''Escherichia coli''. MARINE BIOTECHNOLOGY(2005) 7, 515-522
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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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[5]Seon-Kang C, et al. Characterization of bacterial β-carotene 3,3′-hydroxylases, CrtZ,
 +
and P450 in astaxanthin biosynthetic pathway and adonirubin
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production by gene combination in ''Escherichia coli''. Appl Microbiol Biotechnol (2006) 72
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[6]Nishizaki T, et al. Metabolic engineering of carotenoid biosynthesis in ''Escherichia coli'' by ordered gene assembly in Bacillus subtilis. Appl Environ Microbiol. 2007 Feb
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[[Image:tokyotech_Fig. 1.1.7.1.BBa_K395704.jpg|right|300px|Fig. 1.1.7.BBa_K395704]]
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[7]Arunkumar K, et al. Structure of a carotenoid gene cluster from Pantoea sp. strain C1B1Y and characterization of β-carotene hydroxylase(crtZ)gene by functional complementation in ''Escherichia coli''. Carotenoid Science Vol.11 (2007)
 +
==appendix==
 +
===&beta;-carotene synthesis===
 +
[[Image:tokyotech_Fig. 1.1.2.1. &beta;-carotene.jpg|right|200px|thumb|Fig.2-1-4 &beta;-carotene]]
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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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[[Image:tokyotech_Fig. 1.1.8.1. zeaxanthin pellet and plate.jpg|left|300px|Fig. 1.1.8.1. zeaxanthin pellet and plate]]
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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 DH5&alpha; respectively.  
 +
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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[[Image:tokyotech_table. 1.1.1.2.jpg|500px|center|thumb|table<1>]]
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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 S1], [https://2010.igem.org/Image:Tokyotech_Fig._2-1-10_1BBa_K395706.jpg S2])
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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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~acetone extract~
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[https://2010.igem.org/Team:Tokyo_Tech/Project/Apple_Reporter#Materials_and_Methods &rarr;protocol]
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From the result, we decided to use pBad/araC promoter and double plasimds construct.
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[[Image:tokyotech_Fig. 1.1.3. &beta;-carotene extract.jpg|300px|left|thumb|Fig.2-1-10. &beta;-carotene extract      /worked by Yumiko Kinoshita]]
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==fragrance==
 
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=overview=
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[[Image:tokyotech_Fig. 1.1.4. &beta;-carotene plate.jpg|300px|right|thumb|Fig.2-1-11. &beta;-carotene plate        /worked by Yumiko Kinoshita]]
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Apple fragrance is featured by combinations of volatile compounds, including alcohols, aldehydes, ketones, sesquiterpenes and esters. Esters are associated with fruity attributes of fruit fragrance. In the commercial apple cultivar, over 30 esters have been identified.[Ref]The last step in ester biosynthesis is catalyzed by alcohol acyl transferases (AATs) that use coenzyme A(CoA) donors together with alcohol acceptors as substrates.
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We learned it’s able to make E. coli produce apple fragrance by adding the gene MpAAT1 because Acetyl CoA exists originally in E.coli. MpAAT1 produces a predicted protein containing character of other plant acyl transferases. In contrast with production of pigment, production of fragrance has hardly been worked in the iGEM and few parts exist.For these reasons, we introduced a new BioBrick part to synthesize components of apple fragrance.
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=Highlight=
 
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We designed apple fragrance expression system with MpAAT1. Fig. 1.2.1 shows the outline of the system. MpAAT1 converts substrate and Acetyl CoA into apple fragrance.　Acetyl CoA exists originally in E.coli cell.When 2-methyl butanol or butanol is added as substrates, 2-methylbutyl acetate or butyl acetate is produced and it gives off apple fragrance.
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[[Image:Βカロテン２.png|200px|center|thumb|Fig.2-1-12. &beta;-carotene table        /worked by Yumiko Kinoshita]]
 +
==Materials and Methods==
 +
===The work flow to TLC from culture[https://2010.igem.org/Team:Tokyo_Tech/Project/Apple_Reporter#Reference],[https://2010.igem.org/Team:Tokyo_Tech/Project/Apple_Reporter#Reference]===
 +
====Material====
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''E. coli''      : strain MG1655
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 +
reagent      : acetone, methanol, chloroform, hexane
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[[image:Tokyotech apple fregrance 1.png|630px|center|thumb|Fig. 1.2.1.  Apple fragrance expression system]]
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TLC plate      : silicagel 60F<sub>254</sub>
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We performed gas chromatography to confirm the production of the esters, and the results revealed that MpAAT1 successfully converted alcohol and Acetyl CoA into our target esters.
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====culture====
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1. The cells were cultured over night.
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2. 50ul bacterial solution was taken from culture prepared in 1 and moved into 50ml LB culture with appropriate antibiotic. Inducer was added, if necessary.
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3. The cells were incubated at 37°C, 24hour.
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=Result=
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====gathering ''E. coli'' and extract pigment====
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4. After the O.D reach 2.0, culture was centrifuged for 10minutes at
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4°C, 6000rpm,10min
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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 transformed MpAAT1(BBa_K395602) on pSB6 along with pTrx6 into E.coli BL21 DE3, and cultured after addition of alcohols(2-MeBuOH or BuOH) as substrates.
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6. Moved into 2ml tube by using P1000 pipetter at least twice.
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After 12 hours of incubation, we extracted organic soution layer from the culture and analyzed by gas chromatography (Fig. 1.2.2). Peaks of the esters producing apple fragrance(2-MeBuOAc or BuOAc) were detected.   
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7. Solution is centrifuged for 20minutes at 4°C, 14000rpm
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8. Supernatant was removed completely using pipetter. About 100ul of pellet is obtained.
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[[image:tokyotech apple fragrance 2-1.png|630px|left|thumb|Fig. 1.2.2.  Gas chromatography analysis of apple fragrance expression system.]]
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9. Pellet is added with 500ul acetone, and vortexed for 10 minutes to extract the pigment.
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We designed and constructed a system for apple fragrance production from intracellular metabolites (BBa_K395602). We analyzed cultures with the apple fragrance biosynthetic system for Butyl acetate and 2-methylbutyl acetate production and measured by gas chromatography.(A)Apple fragrance generator (BBa_K395602) in the presence of butanol produces measurable quantities of butyl acetate.(B)Apple fragrance generator (BBa_K395602) does not produce measurable quantities of butyl acetate in the absence of butanol.(C)For reference, we analyzed pure butyl acetate.(D)Apple fragrance generator (BBa_K395602) in the presence of 2-methyl butanol produces measurable quantities of 2-methylbutyl acetate.(E)Apple fragrance generator (BBa_K395602) in the absence of 2-methyl butanol does not produce measurable quantities of 2-methylbutyl acetate(F)For reference, we analyzed pure 2-metylbutyl acetate.(G)For negative control, we analyzed with no sample. Undecane was used as an internal standard for all samples.
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10. Solution is centrifuged for 20minutes at 4°C, 14000rpm
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=Discussion=
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11. Acetone solution was replaced into 2 ml tube.
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====vacuum and concentrate====
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12. Acetone was removed using rotary evaporator.
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From the result of the experiment above, we can conclude that engineered E.coli successfully converted alcohols added as substrates into esters.
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13. 50 ul of Chloroform/Methanol(weight ratio of 9:1) solution was added.
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Moreover, we synthesized MpAAT1 and esters using E.coli BL21 DE3 as a chassis, which was reported to be implausible in former report(s) (C.R. Shen, J.C. Liao. 2008).
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In 2008, C.R. Shen and J.C. Liao succeeded in synthesizing butanol from E.coli. If we take advantage of this engineered E.coli, we could produce apple fragrance ester without the addition of substrate.
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14. Sample was vortexed and spinned down.
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<!--　ここまでね-->
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15. The water layer was removed.
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</div> <!-- end SubWrapper -->
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====TLC====
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16.  Spot the sample onto TLC silica gel plate, and developed by acetone/hexane(weight ratio of 1:3) solution.
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</div> <!-- end Super_main_wrapper -->
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17. Spot was observed under visible light.

Latest revision as of 03:57, 28 October 2010

iGEM Tokyo Tech 2010 "E.coli with Humanity"

2-1 Color

Contents

Abstract

Our team synthesized β-carotene, zeaxanthin and astaxanthin under several conditions (strain, plasmid copy number,…etc) to confirm the functions of BioBrick parts designed by previous iGEM teams. 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 to produce zeaxanthin, 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.


Fig. 2-1-1. carotenoid synthetic pathway
Fig.2-1-2.TLC worked by Yumiko Kinoshita


→our BioBrick parts [http://partsregistry.org/Part:BBa_K395704 →BBa_395704] [http://partsregistry.org/Part:BBa_K395706 →BBa_395706]

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 wide range of functions. (For example, acting as antioxidants[1], and as precursors to other organic compounds.) Among carotenoids, astaxanthin is not known to be converted to Vitamin A, which is converted from most of carotenoids and is toxic when its concentration too high.

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.

Fig. 2-1-3. note

Results

β-carotene synthesis

Fig.2-1-4.β-carotene

Our team synthesized β-carotene under several conditions to confirm the functions of BioBrick parts designed by Cambridge 2009. (we call it "crtEBIY plasmids".) (See more...)

zeaxanthin synthesis

Fig.2-1-5zeaxanthin

Our team tried to synthesize zeaxanthin by using the constructed ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K395704 BBa_K395704])part to confirm the activity of BioBrick parts designed by Cambredge 2009 and Edinburgh 2007. We also confirmed zeaxanthin by TLC (Fig.2-1-6 spot III).


By introduction of crtZ gene to the crtEBIY plasmid (we call this construct “single plasmid construct”, "crtZ-crtEBIY".), new spot appered on TLC plate (Fig.2-1-6 spot III). The spot shows lower Rf value than that derived from crtEBIY (Fig.2-1-6 spot II). The Rf values of spots derived from crtEBIY and crtZ-crtEBIY corresponded to reported values of β-caroten and Zeaxanthin, respectively.[2] Thus we could characterize zeaxanthin production activity of crtZ. In addition, caracterization of the part crtZ is the first work in iGEM.


In order to maximize zeaxanthin production, we designed some combinations of construct. We thought of another type of zeaxanthin synthetic construct where “crtZ” (BBa_I742158) and “crtEBIY” (BBa_K274210) were separated on different plasmids. (we call this type of constructs “double plasmids construct.”)


After all, We made five combinations shown in table<1>, including [http://partsregistry.org/wiki/index.php?title=Part:BBa_K395701 BBa_K395701], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K395702 BBa_K395702] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K395704 BBa_K395704].


Constructs containing pBad/araC promoter on high-copy vector show the best production and double plasmids construct shows as much production as single plasmid construct under same promoter combination. 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.）


For TLC, We selected the best three samples including the sample derived from single plasmid construct .

In the consequence of TLC, we could confirm that these three samples were zeaxanthin.



S1 : zeaxanthin expression components
Fig.2-1-6 zeaxanthin TLC worked by Yumiko Kinoshita
Fig.2-1-7 table<1>　 zeaxanthin worked by Yumiko Kinoshita

astaxanthin synthesis

Fig.2-1-8 astaxanthin

Our team synthesized astaxanthin for the first time in iGEM. Thus the carotenoid synthetic pathway reached the end. This work was done by assembling the new BioBrick part crtW and zeaxanthin synthesis constructs we designed. From the result of “zeaxanthin synthesis”, we decided to use pBad/araC promoter and double plasmids construct. After constructing the new BioBrick device ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K395706 BBa_K395706]) to synthesize astaxanthin, we characterize the function under the condition I in table<2>. Thus we confirmed astaxanthin by TLC (Fig2-1-6 spot IV).


By introduction of crtW gene to the crtZ plasmid (the second construct of five combinations in “zeaxanthin synthesis”,having pBad/araC promoter), new spot ((Fig2-1-6 spot IV) appeared on TLC plate. The spot shows lower Rf value than that derived from crtEBIY (II) and a little higher Rf value than that derived from crtZ-crtW(III). All the Rf values of spots corresponded to reported values respectively.[2] Thus we succeeded in characterizing astaxanthin production activity of crtW. ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K395705 BBa_K395705], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K395706 BBa_K395706])


For the same reason as “zeaxanthin synthesis”, we examined under several conditions →table<2>.

In the consequence of TLC, we could confirm that these four samples were astaxanthin.


Not only astaxanthin but also another spots were produced at II.

We regarded them as intermediary metabolites between zeaxanthin and astaxanthin.



The activity of pBad/araC promoter is measured in detail on another page. →click


S2 : astaxanthin expression component
Fig.2-1-6 TLC astaxanthin worked by Yumiko Kinoshita
Fig.2-1-9 astaxanthin worked by Yumiko Kinoshita


acetone extract and vacuum concentration →protocol

Reference

[1] Yousry M. Antioxidant Activities of Astaxanthin and Related Carotenoids. J. Agric. Food Chem. 2000, 48, 1150-1154 [2]N.M. Sachindra, et al. Carotenoids in crabs from marine and fresh waters of India. LWT 38 (2005) 221–225

[3]MISAWA N, et al. Structure and Functional Analysis of a Marine Bacterial Carotenoid Biosynthesis Gene Cluster and Astaxanthin Biosynthetic Pathway Proposed at the Gene Level. JOURNAL OF BACTERIOLOGY, Nov. 1995, p. 6575–6584 Vol. 177, No. 22

[4]Seon-Kang C, et al. Characterization of bacterial β-carotene Ketolases, CrtW, from Mrine Bacteria by Complementation Analysis in Escherichia coli. MARINE BIOTECHNOLOGY(2005) 7, 515-522

[5]Seon-Kang C, et al. Characterization of bacterial β-carotene 3,3′-hydroxylases, CrtZ, and P450 in astaxanthin biosynthetic pathway and adonirubin production by gene combination in Escherichia coli. Appl Microbiol Biotechnol (2006) 72


[6]Nishizaki T, et al. Metabolic engineering of carotenoid biosynthesis in Escherichia coli by ordered gene assembly in Bacillus subtilis. Appl Environ Microbiol. 2007 Feb

[7]Arunkumar K, et al. Structure of a carotenoid gene cluster from Pantoea sp. strain C1B1Y and characterization of β-carotene hydroxylase(crtZ)gene by functional complementation in Escherichia coli. Carotenoid Science Vol.11 (2007)

appendix

β-carotene synthesis

Fig.2-1-4 β-carotene

Our team synthesized β-carotene under several conditions to confirm the activity of BioBrick parts designed by Cambredge 2009.

We used crtEBIY (BBa_K274210) to synthesize β-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.

We found that MG1655 and JM109 produce more β-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.

We also made special competent cell, MG1655 and JM109, having a plasmid crtEBIY; pSB1A2 or crtEBIY; pSB3K3) for zeaxanthin and astaxanthin synthesis.(→S1, S2)


~acetone extract~ →protocol

Fig.2-1-10. β-carotene extract /worked by Yumiko Kinoshita


Fig.2-1-11. β-carotene plate /worked by Yumiko Kinoshita


Fig.2-1-12. β-carotene table /worked by Yumiko Kinoshita

Materials and Methods

The work flow to TLC from culture[3],[4]

Material

E. coli  : strain MG1655

reagent  : acetone, methanol, chloroform, hexane

TLC plate  : silicagel 60F254

culture

1. The cells were cultured over night.

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

3. The cells were incubated at 37°C, 24hour.

gathering E. coli and extract pigment

4. After the O.D reach 2.0, culture was centrifuged for 10minutes at 4°C, 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°C, 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°C, 14000rpm

11. Acetone solution was replaced into 2 ml tube.

vacuum and concentrate

12. Acetone was removed using rotary evaporator.

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

14. Sample was vortexed and spinned down.

15. The water layer was removed.

TLC

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

17. Spot was observed under visible light.
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