Coding
"The Gun"

Part:BBa_K1080003

Designed by: Macquarie University   Group: iGEM13_Macquarie_Australia   (2013-09-12)

GUN4


Description & Function


GUN4 (Genomes uncoupled 4) is a protein which is found only in aerobic photosynthetic organisms. It is a tetrapyrrole-binding protein which controls levels of magnesium-protoporhyrin IX which is a crucial component for the production of chlorophyll within photosynthetic organisms. GUN4 is thought to participate in plastid-to-nucleus signalling by regulating magnesium-protoporphyrin IX synthesis or trafficking

Operon usage


PSB1C3 Operon 1.png
Figure 1 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.

Biobrick Design:

Source Genbank accession: [http://www.ncbi.nlm.nih.gov/nuccore/NW_001843867.1?report=genbank&from=971974&to=974471 NW_001843867]

Source Uniprot reference: [http://www.uniprot.org/uniprot/A8I5N5 A8I5N5]

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 1 synthesised DNA fragment and 1 PCR fragment into BB vector.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 719
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 404
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]



Experimental Validation

GUN4 expressed cells were lysed and the protein precipitated by methanol. The supernatant was then loaded onto a [http://2014.igem.org/Team:Macquarie_Australia/WetLab/Protocols/Operons UHPLC] column (reverse phase) to separate organic compounds [2]. The target compound protoporphyrin-IX is expected to elute at 11.1 mins, based on a commercial protoporphyrin-IX standard (figure 3).

Negative Control HPLC Trace.jpg
Figure 2 UHPLC trace of methanol solvent only. No peak at 11.1 mins observed.

Positive Control Protoporphyrin IX HPLC Trace.jpg
Figure 3 UHPLC trace of Protoporphyrin IX standard. Note peak intensity at 11.1 mins consistent with Protoporphyrin IX.

GUN4 HPLC Trace.jpg
Figure 4 UHPLC trace of organic compounds from GUN4 expressed cell lysate. Protoporphyrin-IX peak is at 11.1 mins, with a peak area of 29.

YCF54 Only HPLC.jpg
Figure 5 HPLC trace for YCF54 expressed cells (control) . Protoporphyrin IX peak is at 11.1 mins, with a peak area of 158.





Amino acid sequence

MAMRVTVAAG KLDSVSLFGG DTASLMGGSQ TVEKKKSGKE AVMEVQLSST AGIDYTVLRD
HLANGEFREA EDETRALLIK LAGPEAVKRN WVYFTEVKNI SVTDFQTLDN LWKASSNNKF
GYSVQKEIWV QNQKRWPKFF KQIDWTQGEN NNYRKWPMEF IYSMDAPRGH LPLTNALRGT
QLFQAIMEHP AFEKSSTAKT LDQKAAEAAG RTQSLF

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


Number of amino acids: 216

Molecular weight: 24350.6

Theoretical pI: 8.98


Amino acid composition:

Ala(A) 21 ( 9.7% )
Arg(R) 10 ( 4.6% )
Asn(N) 11 ( 5.1% )
Asp(D) 10 ( 4.6% )
Cys(C) 00 ( 0.0% )
Gln(Q) 12 ( 5.6% )
Glu(E) 15 ( 6.9% )
Gly(G) 14 ( 6.5% )
His(H) 03 ( 1.4% )
Ile(I) 07 ( 3.2% )
Leu(L) 17 ( 7.9% )
Lys(K) 18 ( 8.3% )
Met(M) 07 ( 3.2% )
Phe(F) 11 ( 5.1% )
Pro(P) 06 ( 2.8% )
Ser(S) 15 ( 6.9% )
Thr(T) 15 ( 6.9% )
Trp(W) 06 ( 2.8% )
Tyr(Y) 05 ( 2.3% )
Val(V) 13 ( 6.0% )
Pyl(O) 00 ( 0.0% )
Sec(U) 00 ( 0.0% )

(B)   0	  0.0%
(Z)   0	  0.0%
(X)   0	  0.0%


Total number of negatively charged residues (Asp + Glu): 25 Total number of positively charged residues (Arg + Lys): 28


Atomic composition:

Carbon C 1085 Hydrogen H 1693 Nitrogen N 299 Oxygen O 325 Sulfur S 7

Formula: C1085H1693N299O325S7 Total number of atoms: 3409

Extinction coefficients:

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

Ext. coefficient 40450 Abs 0.1% (=1 g/l) 1.661


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 22.44 This classifies the protein as stable.


Aliphatic index: 70.51

Grand average of hydropathicity (GRAVY): -0.508


Ribbon structure of protein

http://pfam.sanger.ac.uk/structure/getimage?id=1y6i

Source

Chlamydomonas reinhardtii

References

[1] Brzezowski, P., et al. (2014). "The GUN4 protein plays a regulatory role in tetrapyrrole biosynthesis and chloroplast-to-nucleus signalling in Chlamydomonas reinhardtii." Plant J. 79(2): 285-98
[2] Benton, C. M., Lim, C. K., Moniz, C., & Jones, D. J. (2012). Ultra high-performance liquid chromatography of porphyrins in clinical materials: column and mobile phase selection and optimisation. Biomed Chromatogr, 26(6), 714-719. doi: 10.1002/bmc.1720


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