Part:BBa_K1080009
ChlG
Chlorophyll synthetase - Catalyses the esterification of chlorophyllide with phytyl-pyrophosphate or geranyl-geranyl pyrophosphate in chloroplasts which finalizes the production of chlorophyll. ChlG expression and activity heavily impacts on a plants capacity to photosynthesize.
Operon usage
This gene has been used in an operon with other genes responsible for the terminal steps of the chlorophyll biosynthesis pathway, in the conversion of divinyl protochlorophyllide to chlorophyll a. DVR1 reduces divinyl protochlorophyllide, POR converts protochlorophyllide to chlorophyllide, ChlG adds the geranylgeranyl pyrophosphate chain to the chlorophyllide molecule, and ChlP reduces the double bonds on GGPP. The final product is chlorophyll a.
The plasmid is under the control of the lac promoter.
Biobrick Design:
Source Genbank accession: [http://www.ncbi.nlm.nih.gov/nuccore/NW_001843885.1?report=genbank&from=78801&to=82699&strand=true NW_001843885.1]
Source Uniprot reference: [http://www.uniprot.org/uniprot/A8JFJ1 A8JFJ1]
cDNA gene sequence from Chlamydomonas reinhardtii was sourced from NCBI database, chloroplast targeting sequence was removed. EcoRI/XbaI/SpeI/PstI restriction sites were removed via codon adjustment, biobrick prefix and RBS were added to start of gene, biobrick suffix added to end of gene.
Biobrick construction: Gibson assembly of 2 synthesised DNA fragments into BB vector.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 386
Illegal BglII site found at 875
Illegal BamHI site found at 155 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Amino acid sequence
MNQQATEEKS DTNSAARQML GMKGAALETD IWKIRVQLTK PVTWIPLIWG VACGAAASGH YQWNNPTQIA QLLTCMMMSG PFLTGYTQTI NDWYDREIDA INEPYRPIPS GRISERDVIV QIWVLLLGGI GLAYTLDQWA GHTTPVMLQL TIFGSFISYI YSAPPLKLKQ SGWAGNYALG SSYIALPWWA GQALFGTLTL DVMALTIAYS LAGLGIAIVN DFKSIEGDRQ MGLQSLPVAF GVDTAKWICV STIDVTQLGV AAYLAWGLHE ELYGAVLLAL ILPQIYFQYK YFLPDPIAND VKYQASAQPF LVFGLLTAGL ACGHHVNAVA AAASAAGAL
References and documentation are available. Please note the modified algorithm for extinction coefficient.
Number of amino acids: 339
Molecular weight: 36830.6
Theoretical pI: 5.30
Amino acid composition:
Ala (A) 43 12.7%
Arg (R) 7 2.1%
Asn (N) 10 2.9%
Asp (D) 14 4.1%
Cys (C) 4 1.2%
Gln (Q) 20 5.9%
Glu (E) 9 2.7%
Gly (G) 30 8.8%
His (H) 5 1.5%
Ile (I) 26 7.7%
Leu (L) 40 11.8%
Lys (K) 10 2.9%
Met (M) 9 2.7%
Phe (F) 10 2.9%
Pro (P) 16 4.7%
Ser (S) 18 5.3%
Thr (T) 21 6.2%
Trp (W) 12 3.5%
Tyr (Y) 16 4.7%
Val (V) 19 5.6%
Pyl (O) 0 0.0%
Sec (U) 0 0.0%
(B) 0 0.0% (Z) 0 0.0% (X) 0 0.0%
Total number of negatively charged residues (Asp + Glu): 23
Total number of positively charged residues (Arg + Lys): 17
Atomic composition:
Carbon C 1694 Hydrogen H 2600 Nitrogen N 422 Oxygen O 471 Sulfur S 13
Formula: C1694H2600N422O471S13 Total number of atoms: 5200
Extinction coefficients:
Extinction coefficients are in units of M-1 cm-1, at 280 nm measured in water.
Ext. coefficient 90090 Abs 0.1% (=1 g/l) 2.446, assuming all pairs of Cys residues form cystines
Ext. coefficient 89840
Abs 0.1% (=1 g/l) 2.439, assuming all Cys residues are reduced
Estimated half-life:
The N-terminal of the sequence considered is M (Met).
The estimated half-life is:
30 hours (mammalian reticulocytes, in vitro).
>20 hours (yeast, in vivo).
>10 hours (Escherichia coli, in vivo).
Instability index:
The instability index (II) is computed to be 26.91 This classifies the protein as stable.
Aliphatic index: 104.87
Grand average of hydropathicity (GRAVY): 0.327
Source
Chlamydomonas reinhardtii
References
Nikolai, S., et al. (2009). "Expression of chlorophyll synthase is also involved in feedback-control of chlorophyll biosynthesis." An international journal on molecular biology, molecular genetics and biochemistry.
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