Coding
"D"

Part:BBa_K1080002

Designed by: Macquarie University   Group: iGEM13_Macquarie_Australia   (2013-09-22)
Revision as of 09:59, 17 October 2014 by Leah.Simmons (Talk | contribs)

ChlD

Magnesium chelatase subunit D

Forms an ATP dependent complex with the ChlI subunits 1 & 2, before acting on the protoporphyrin which is bound to the ChlH protein to insert magnesium.

Operon usage


PSB1C3 Operon 1.png
This gene has been used in an operon with other genes responsible for the synthesis pathway from Protoporphyrin IX to MG-protoporphyrin IX. ChlI1 and ChlD form an Mg-chelatase complex, which acts upon protoporphyrin, catalysing the insertion of a Magnesium ion into the centre of the protoporphyrin IX. GUN4 aids this reaction by activating the Mg-chelatase enzyme complex.
The plasmid is under the control of the lac promoter.
Future plans involve adding ChlH to this operon, which binds Mg-chelatase with GUN4 to protoporphyrin.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 1334
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 2039
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 614
    Illegal NgoMIV site found at 1433
    Illegal NgoMIV site found at 1975
  • 1000
    COMPATIBLE WITH RFC[1000]


Characterisation of ChlD


A simple assay is available to experimentally validate the function of our ChlD Biobrick. The basis of this test is to provide all proteins required to form the Magnesium Chelatase complex, except for ChlD. When the ChlD protein is present, Magnesium-protoporphyrin is formed from protoporphyrin already present in the E. coli. This formation can be observed via fluorescence, and we have performed this assay with the same setup as used in Zhou et al, 2010:

The final concentrations of assay components were 50 mM Tricine–NaOH (pH 8.0), 15 mM MgCl2, 2 mM dithiothreitol (DTT), 1 mM ATP (assay buffer), 2000 nM Proto, 200 nM CrChlI1 and 200nM CrChlI2, 500 nM CrChlH, 500 nM CrGUN4. ChlD Extract was added to CrI1 and CrI2 at 2x assay concentration in reaction buffer. Assay was started by adding 25 uL of Cr-ChlH and Cr-GUN4 at 2x assay concentration to 25 uL of the Cr-I1 Cr-I2 ChlD extract in assay buffer.

ChlD expressing E. coli cells were lysed with B-PER bacterial cell lysis reagent (Pierce) as per instructions. 2 ul of this extract gave the equivalent of 2.1ng of purified ChlD activity when added to the assay. The B-PER protein concentration was approximately 10mg/ml, indicating a low expression of the gene. Improvements of expression level may be obtained by using alternate promoters. The control used was a ChlI1 expressing cell extract at the same concentration (ie with ChlD activity). Assay was monitored every 2 seconds for 100 seconds using fluorescence detection in a Pherastar plate reader with a 420nm excitation filter and a 590nm emission filter.

Graph 1 shows the fluorescence tracking for our control E. coli (without ChlD activity):



Graph 2 shows the fluorescence of two of our ChlD subcultures, with increasing fluorescence detected, indicating Magnesium-protoporphyrin-IX formation. Red line shows ChlD2 activity, green line shows ChlD4 activity:



References:

Zhou, S., Sawicki, A., Willows, R. D., & Luo, M. (2012). C-terminal residues of Oryza sativa GUN4 are required for the activation of the ChlH subunit of magnesium chelatase in chlorophyll synthesis. FEBS letters, 586(3), 205-210.

Amino acid sequence:

MRAMKVSEED SKGFDADVST RLARSYPLAA VVGQDNIKQA LLLGAVDTGL GGIAIAGRRG
TAKSIMARGL HALLPPIEVV EGSICNADPE DPRSWEAGLA EKYAGGPVKT KMRSAPFVQI
DGVNVVEREG ISISHPCRPL LIATYNPEEG PLREHLLDRI AIGLSADVPS TSDERVKAID
AAIRFQDKPQ DTIDDTAELT DALRTSVILA REYLKDVTIA PEQVTYIVEE ARRGGVQGHR
AELYAVKCAK ACAALEGRER VNKDDLRQAV QLVILPRATI LDQPPPEQEQ PPPPPPPPPP
PPPQDQMEDE DQEEKEDEKE EEEKENEDQD EPEIPQEFMF ESEGVIMDPS ILMFAQQQQR
AQGRSGRAKT LIFSDDRGRY IKPMLPKGDK VKRLAVDATL RAAAPYQKIR RQQAISEGKV
QRKVYVDKPD MRSKKLARKA GALVIFVVDA SGSMALNRMS AAKGACMRLL AESYTSRDQV
VMMVLITDGR ANVSLAKSNE DPEALKPDAP KPTADSLKDE VRDMAKKAAS AGINVLVIDT
ENKFVSTGFA EEISKAAQGK YYYLPNASDA AIAAAASGAM AAAKGGY

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

Number of amino acids: 707

Molecular weight: 76420.1

Theoretical pI: 5.23

Amino acid composition:
Ala (A) 88 12.4%
Arg (R) 47 6.6%
Asn (N) 14 2.0%
Asp (D) 51 7.2%
Cys (C) 7 1.0%
Gln (Q) 31 4.4%
Glu (E) 55 7.8%
Gly (G) 51 7.2%
His (H) 6 0.8%
Ile (I) 40 5.7%
Leu (L) 60 8.5%
Lys (K) 43 6.1%
Met (M) 20 2.8%
Phe (F) 12 1.7%
Pro (P) 50 7.1%
Ser (S) 42 5.9%
Thr (T) 24 3.4%
Trp (W) 1 0.1%
Tyr (Y) 16 2.3%
Val (V) 49 6.9%
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): 106 Total number of positively charged residues (Arg + Lys): 90

Atomic composition:

Carbon C 3333 Hydrogen H 5435 Nitrogen N 949 Oxygen O 1047 Sulfur S 27

Formula: C3333H5435N949O1047S27 Total number of atoms: 10791

Extinction coefficients:

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

Ext. coefficient 29715 Abs 0.1% (=1 g/l) 0.389, assuming all pairs of Cys residues form cystines


Ext. coefficient 29340 Abs 0.1% (=1 g/l) 0.384, 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 45.56 This classifies the protein as unstable.


Aliphatic index: 87.71

Grand average of hydropathicity (GRAVY): -0.337

Source

Chlamydomonas reinhardtii


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