Difference between revisions of "Part:BBa K1080010"

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The structure of the chlorophyll molecule is the isocyclic ring. The formation of the ring is dependent by the action of enzyme Mg-protoporphyrin IX monomethyl ester (oxidative) cyclase. The catalysis of this enzyme to divinyl protochlorophyllide.  
 
The structure of the chlorophyll molecule is the isocyclic ring. The formation of the ring is dependent by the action of enzyme Mg-protoporphyrin IX monomethyl ester (oxidative) cyclase. The catalysis of this enzyme to divinyl protochlorophyllide.  
 
Essential for the formation of protochlorophyllide is the catalysis of Protoporphyrin Monomethylester Oxidative Cyclase. This is achieved by the interaction YCF 54 with other components integral in the formation, XanL or CTH1 and plastocyanin. The activity and stability of the Protoporphyrin Monomethylester Oxidative Cyclase is dependent on the YCF54 component when forming protochlorophyllide.   
 
Essential for the formation of protochlorophyllide is the catalysis of Protoporphyrin Monomethylester Oxidative Cyclase. This is achieved by the interaction YCF 54 with other components integral in the formation, XanL or CTH1 and plastocyanin. The activity and stability of the Protoporphyrin Monomethylester Oxidative Cyclase is dependent on the YCF54 component when forming protochlorophyllide.   
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===Operon Usage===
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[[File:pSB1C3_Operon 2.png]]
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<br>This gene has been used in an operon with other genes responsible for catalysing the biosynthesis pathway from Mg-protoporphyrin IX to Protochlorophyllide. [https://parts.igem.org/wiki/index.php?title=Part:BBa_K1080005 CTH1], [https://parts.igem.org/Part:BBa_K1080006 Plastocyanin], and [https://parts.igem.org/wiki/index.php?title=Part:BBa_K1080010 YCF54] are involved in the oxidative cyclase pathway. [https://parts.igem.org/Part:BBa_K1080004 ChlM] methylates Mg-protoporphyrin IX, facilitating the highly-regulated catalysis of Mg-chelatase. [https://parts.igem.org/wiki/index.php?title=Part:BBa_K1080005 CTH1] catalyses the conversion of Mg-protoporphyrin IX monomethyl into divinyl protochlorophyllide, interacting with [https://parts.igem.org/wiki/index.php?title=Part:BBa_K1080010 YCF54] and [https://parts.igem.org/Part:BBa_K1080006 Plastocyanin].
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<br>The insertion of magnesium is the key component of the chlorophyll biosynthesis pathway.
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<br>The plasmid is under the control of the [https://parts.igem.org/Part:BBa_K864400 lac promoter].
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[[File:The structure of the chlorophyll molecule is the isocyclic ring. The formation of the ring is dependent by the action of enzyme Mg-protoporphyrin IX monomethyl ester (oxidative) cyclase. The catalysis of this enzyme to divinyl protochlorophyllide.png]]
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<b><h4>Biobrick Design:</h4></b>
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Source Genbank accession: [http://www.ncbi.nlm.nih.gov/nuccore/NW_001843510.1?report=genbank&from=2030539&to=2032329&strand=true NW_001843510.1]
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Source Uniprot reference: [http://www.uniprot.org/uniprot/A8ILP2 A8ILP2]
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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.
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<b>Biobrick construction:</b> Restriction and ligation of 1 synthesised DNA fragment into BB vector.
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===References===
 
===References===
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Hollingshead, S., et al. (2012). "Conserved chloroplast open-reading frame ycf54 is required for activity of the magnesium protoporphyrin monomethylester oxidative cyclase in Synechocystis PCC 6803." J Biol Chem 287(33): 27823-27833.
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Latest revision as of 17:47, 16 October 2014

YCF54

Usage and Biology

The structure of the chlorophyll molecule is the isocyclic ring. The formation of the ring is dependent by the action of enzyme Mg-protoporphyrin IX monomethyl ester (oxidative) cyclase. The catalysis of this enzyme to divinyl protochlorophyllide. Essential for the formation of protochlorophyllide is the catalysis of Protoporphyrin Monomethylester Oxidative Cyclase. This is achieved by the interaction YCF 54 with other components integral in the formation, XanL or CTH1 and plastocyanin. The activity and stability of the Protoporphyrin Monomethylester Oxidative Cyclase is dependent on the YCF54 component when forming protochlorophyllide.

Operon Usage

PSB1C3 Operon 2.png
This gene has been used in an operon with other genes responsible for catalysing the biosynthesis pathway from Mg-protoporphyrin IX to Protochlorophyllide. CTH1, Plastocyanin, and YCF54 are involved in the oxidative cyclase pathway. ChlM methylates Mg-protoporphyrin IX, facilitating the highly-regulated catalysis of Mg-chelatase. CTH1 catalyses the conversion of Mg-protoporphyrin IX monomethyl into divinyl protochlorophyllide, interacting with YCF54 and Plastocyanin.
The insertion of magnesium is the key component of the chlorophyll biosynthesis pathway.
The plasmid is under the control of the lac promoter.

Biobrick Design:

Source Genbank accession: [http://www.ncbi.nlm.nih.gov/nuccore/NW_001843510.1?report=genbank&from=2030539&to=2032329&strand=true NW_001843510.1]

Source Uniprot reference: [http://www.uniprot.org/uniprot/A8ILP2 A8ILP2]

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: Restriction and ligation of 1 synthesised DNA fragment into BB vector.

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 108
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 270




Amino acid sequence

MAPAAASADK ATAAEYYALV CNAEWFFMDP QNESVAEQLR EKVRFFKEQN KERDFFIVPN
PKWLDAKFPE QAKQVKRPCV ALVSTDKMWI TFMKLRLDRV LKIDLKSMPA SEVLAAGEAL
PDFKPDGKWT APYARYTPGW WNVFLPNH


References and documentation are available. Please note the modified algorithm for extinction coefficient.

Number of amino acids: 148

Molecular weight: 17073.7

Theoretical pI: 8.50

Amino acid composition:
Ala (A) 20 13.5%
Arg (R) 7 4.7%
Asn (N) 6 4.1%
Asp (D) 9 6.1%
Cys (C) 2 1.4%
Gln (Q) 5 3.4%
Glu (E) 10 6.8%
Gly (G) 3 2.0%
His (H) 1 0.7%
Ile (I) 3 2.0%
Leu (L) 11 7.4%
Lys (K) 14 9.5%
Met (M) 5 3.4%
Phe (F) 10 6.8%
Pro (P) 12 8.1%
Ser (S) 5 3.4%
Thr (T) 5 3.4%
Trp (W) 6 4.1%
Tyr (Y) 4 2.7%
Val (V) 10 6.8%
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): 19 Total number of positively charged residues (Arg + Lys): 21

Atomic composition:

Carbon C 785 Hydrogen H 1190 Nitrogen N 202 Oxygen O 212 Sulfur S 7

Formula: C785H1190N202O212S7 Total number of atoms: 2396

Extinction coefficients:

Extinction coefficients are in units of M-1 cm-1, at 280 nm measured in water.

Ext. coefficient 39085 Abs 0.1% (=1 g/l) 2.289, assuming all pairs of Cys residues form cystines


Ext. coefficient 38960 Abs 0.1% (=1 g/l) 2.282, 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 35.64 This classifies the protein as stable.


Aliphatic index: 70.00

Grand average of hydropathicity (GRAVY): -0.385

Source

Chlamydomonas reinhardtii

References

Hollingshead, S., et al. (2012). "Conserved chloroplast open-reading frame ycf54 is required for activity of the magnesium protoporphyrin monomethylester oxidative cyclase in Synechocystis PCC 6803." J Biol Chem 287(33): 27823-27833.