Difference between revisions of "Part:BBa K118003"

Latest revision as of 07:55, 24 October 2016

crtI coding sequence encoding phytoene dehydrogenase

This is the coding sequence of crtI from Pantoea ananatis (formerly Erwinia uredovora) (Accession number D90087). It encodes phytoene dehydrogenase, part of the carotenoid biosynthesis pathway, which converts Phytoene to Lycopene (Misawa, N., Nakagawa, N., Kobayashi, K., Yamano, S., Nakamura, K., and Harashima, K. 1990. Elucidation of the Erwinia uredovora carotenoid biosynthetic pathway by functional analysis of gene products expressed in Escherichia coli. Journal of Bacteriology 172, 6704-612). Two PstI sites were removed by site-directed mutagenesis.

Structure

Crystal structure of Phytoene desaturase CRTI from Pantoea ananatis

Improvement on BBa_K118003

　BBa_K118003 is the coding sequence of phytoene desaturase (CrtI/PDS) in Erwinia uredovora. CrtI is an enzyme catalyzes the conversion of phytoene to lycopene in a one-step reaction. In iGEM16_SCAU-China project, CrtI is used to simplified a reconstructed pathway to achieve astaxanthin biosynthesis in higher plant, rice (Oryza sativa). The original CrtI is not suitable to express in higher plants. Thus, codon optimization on CrtI had made for rice. In addition, a Pea transit peptide of Rubisco small subunit is fused to the optimized CDS of CrtI for proper sorting into the plastid. Comparison of nucleaotide and amino acid sequence of original CrtI (EuCrtI) and codon-optimized CrtI (coCrtI) are shown in Figure.1 and Figure.2.

Figure. 1　　Sequence difference between EuCrtI (BBa_K118003) and coCrtI (BBa_K1959002).

Figure. 2　　Amino acid sequence of translated DNA of EuCrtI (BBa_K118003) and coCrtI (BBa_K1959002).
Sequence alignment shows that there are no differences between amino acid sequence of original CtrI and codon-optimized CrtI.

　With the help of improved part, iGEM16_SCAU-China had successfully achieved astaxanthin biosynthesis in rice (Figure. 3). Content and purity of astaxanthin was determined by HPLC analysis (Figure. 4)

Figure. 3　　The polished rice phenotype of aSTARice.

Figure. 4　　HPLC chromatogram of methanol extracts from transgenic aSTARice (red line) and wild-type (blue line) rice seeds. HPLC analysis recorded at 480 nm of extracts.

　For more details about the design of optimized CrtI, please refer to https://parts.igem.org/Part:BBa_K1959002. For more details and analyses, please visit the wiki of SCAU-China 2016 (http://2016.igem.org/Team:SCAU-China).
[Edited by Junyu Chen, SCAU-China 2016, 19 October 2016(UTC)]

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 71
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

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 <!-- Add more about the biology of this part here
 ===Usage and Biology===
+<!-- -->
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 [[File:BBa K118003-Evry2016.jpeg|frame|left|Crystal structure of Phytoene desaturase CRTI from Pantoea ananatis]]
+<br clear=all>
 ===Improvement on BBa_K118003===
-　BBa_K118003 is the coding sequence of phytoene desaturase (CrtI/PDS) in ''Erwinia uredovora''. CrtI is an enzyme catalyzes the conversion of phytoene to lycopene in a one-step reaction. In iGEM16_SCAU-China project, ''CrtI'' is used to simplified a reconstructed pathway to achieve astaxanthin biosynthesis in higher plant, rice (''Oryza sativa''). The original ''CrtI'' is not suitable to express in higher plants. Thus, codon optimization on ''CrtI'' had made for rice. In addition, a ''Pea'' transit peptide of Rubisco small subunit is fused to the optimized CDS of ''CrtI'' for proper sorting into the plastid. Comparison of nucleaotide and amino acid sequence of original ''CrtI'' (''EuCrtI'') and optimized ''CrtI'' (''OsCrtI'') are shown in Figure.1 and Figure.2.
+　BBa_K118003 is the coding sequence of phytoene desaturase (CrtI/PDS) in ''Erwinia uredovora''. CrtI is an enzyme catalyzes the conversion of phytoene to lycopene in a one-step reaction. In iGEM16_SCAU-China project, ''CrtI'' is used to simplified a reconstructed pathway to achieve astaxanthin biosynthesis in higher plant, rice (''Oryza sativa''). The original ''CrtI'' is not suitable to express in higher plants. Thus, codon optimization on ''CrtI'' had made for rice. In addition, a ''Pea'' transit peptide of Rubisco small subunit is fused to the optimized CDS of ''CrtI'' for proper sorting into the plastid. Comparison of nucleaotide and amino acid sequence of original ''CrtI'' (''EuCrtI'') and codon-optimized ''CrtI'' (''coCrtI'') are shown in Figure.1 and Figure.2.
-[[File:T--SCAU-China--CrtICompare.png|500px|thumb|centre|<p>'''Figure. 1　　Sequence difference between ''EuCrtI'' (BBa_K118003) and ''OsCrtI'' (<partinfo>BBa_K1959002</partinfo>).'''</p>]]
+[[File:T--SCAU-China--CrtICompare.png|500px|thumb|centre|<p>'''Figure. 1　　Sequence difference between ''EuCrtI'' (BBa_K118003) and ''coCrtI'' (<partinfo>BBa_K1959002</partinfo>).'''</p>]]
-[[File:T--SCAU-China--CrtIpro.jpg |500px|thumb|centre|<p>'''Figure. 2　　Amino acid sequence of translated DNA of  ''EuCrtI'' (BBa_K118003) and ''OsCrtI'' (<partinfo>BBa_K1959002</partinfo>).'''<br>Sequence alignment shows that there are no differences between amino acid sequence of original ''CtrI'' and codon-optimized ''CrtI''.</p>]]
+[[File:T--SCAU-China--CrtIpro.jpg |500px|thumb|centre|<p>'''Figure. 2　　Amino acid sequence of translated DNA of  ''EuCrtI'' (BBa_K118003) and ''coCrtI'' (<partinfo>BBa_K1959002</partinfo>).'''<br>Sequence alignment shows that there are no differences between amino acid sequence of original ''CtrI'' and codon-optimized ''CrtI''.</p>]]
 　With the help of improved part, iGEM16_SCAU-China had successfully achieved astaxanthin biosynthesis in rice (Figure. 3). Content and purity of astaxanthin was determined by HPLC analysis (Figure. 4)