Difference between revisions of "Part:BBa K2664006"

Revision as of 11:45, 17 October 2018

Mg chelatase plasmid

Sequence and Features

Overview

The fate of the protoporphyrin IX (PPIX) is dependent upon which enzyme is present to convert PPIX to the next product. For instance, ferrochelatase (encoded by hemH) converts PPIX to FePPIX (heme). Our magnesium chelatase plasmid is an assembly of five biobricks, a combination of new and previously registered by past Macquarie iGEM teams. These genes form the first part of the chlorophyll biosynthesis pathway and are found in our plasmid with the following order; ChlI1, ChlI2, ChlD, ChlH and GUN4. They encode for Magnesium chelatase subunits I (ChlI1, ChlI2), D (ChlD) and H (ChlH), as well as for Genomes uncoupled 4 (gUN4). All together, they catalyse the reaction of converting protoporphyrin IX to divinyl protochlorophyllide in the presence of NADPH and O2.
The genes' sequences have been designed to contain a ribosome binding site (AGGAGG), located six codons upstream the start codon for every gene. Additionally, the genes ChlI1 and ChlH carry the trc promoter, instead of the plac promoter that was previously present.

Biology & Literature

The genes ChlI1 and ChlI2 form a Mg-cheltase complex (Magnesium cheltase subunit I). This complex catalyses the insertion of magnesium ion into protoporphyrin IX to yield Mg-protoporphyrin IX by forming two complexes; an ATP dependent hexameric ring complex and a complex with the chlD subunit.
Next comes chlH, which encodes for the magnesium shelters subunit H. This acts as a chloroplast precursor, catalysing the first step in the chlorophyll biosynthesis pathway by insertion of an Mg2+ ion into protoporphyrin IX to generate Mg-protoporphyrin IX. The complete complex then acts on protoporphyrin. The gUN4 gene encodes for the genomes-uncoupled-4-protein, which is a tetrapyrrole-binding protein that controls the production of Mg-protoporhyrin IX.

The chlH and gUN4 gene encoded proteins bind to protoporphyrin IX, forming an activated complex. This behaves as a substrate for the ChlID motor complex, to insert magnesium into the bound Protoporphyrin IX upon ATP hydrolysis. The substrate complex requires μM concentrations of Protoporphyrin IX and chlH for optimal activity, while the assembly of the complete complex requires mM concentrations of Mg2+ and ATP.

Assembly and Design

Each individual biobrick was assembled in the following order using standard assembly:
trc-ChlI1-ChlI2-ChlD-trc-ChlH-GUN4.
The basic biobricks corresponding to the genes were:
[BBa_K2664004], [BBa_K1080011], [BBa_K1080002], [BBa_K2664005] and [BBa_K1080003], respectively. All of these genes code for different subunits of the enzyme Magnesium chelatase.

All genes within this plasmid are sequences obtained from Chlamydomonas reinhardtii and codon optimised to be expressed in Escherichia coli.

Verification and Validation

Fig 1. Agarose gel electrophoresis (0.5% agarose) with GelRed (2 μl/100 ml) showing single (E) and double (E+P) digests of the Mg-Chelatase plasmid (9513bp).

Protein information

chlI1
Mass: 39.96 kDa
Sequence:
MAATEVKAAEGRTEKELGQARPIFPFTAIVGQDEMKLALILNVIDPKIGGVMIMGDRGTGKSTTIRALADLLPEMQVVANDPFNSDPTDPELMSEEVRNR VKAGEQLPVSSKKIPMVDLPLGATEDRVCGTIDIEKALTEGVKAFEPGLLAKANRGILYVDEVNLLDDHLVDVLLDSAASGWNTVEREGISISHPARFIL VGSGNPEEGELRPQLLDRFGMHAQIGTVKDPRLRVQIVSQRSTFDENPAAFRKDYEAGQMALTQRIVDARKLLKQGEVNYDFRVKISQICSDLNVDGIRG DIVTNRAAKALAAFEGRTEVTPEDIYRVIPLCLRHRLRKDPLAEIDDGDRVREIFKQVFGME

chlI2
Mass: 39.56 kDa
Sequence:
MPSTKAAKKP NFPFVKIQGQ EEMKLALLLN VVDPNIGGVL IMGDRGTAKS VAVRALVDML PDIDVVEGDA FNSSPTDPKF MGPDTLQRFR NGEKLPTVRM RTPLVELPLG ATEDRICGTI DIEKALTQGI KAYEPGLLAK ANRGILYVDE VNLLDDGLVD VVLDSSASGL NTVEREGVSI VHPARFIMIG SGNPQEGELR PQLLDRFGMS VNVATLQDTK QRTQLVLDRL AYEADPDAFV DSCKAEQTAL TDKLEAARQR LRSVKISEEL QILISDICSR LDVDGLRGDI VINRAAKALV AFEGRTEVTT NDVERVISGC LNHRLRKDPL DPIDNGTKVA ILFKRMTDPE IMKREEEAKK

chlD
Mass: 63.84 kDa
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

chlH
Mass:144.14 kDA
Sequence:
MCNVATGPRP PMTTFTGGNK GPAKQQVSLD LRDDGAGMFT STSPEMRRVV PDDVKGRVKV KVVYVVLEAQ YQSAISAAVK NINAKNSKVC FEVVGYLLEE LRDQKNLDML KEDVASANIF IGSLIFIEEL AEKIVEAVSP LREKLDACLI FPSMPAVMKL NKLGTFSMAQ LGQSKSVFSE FIKSARKNND NFEEGLLKLV RTLPKVLKYL PSDKAQDAKN FVNSLQYWLG GNSDNLENLL LNTVSNYVPA LKGVDFSVAE PTAYPDVGIW HPLASGMYED LKEYLNWYDT RKDMVFAKDA PVIGLVLQRS HLVTGDEGHY SGVVAELESR GAKVIPVFAG GLDFSAPVKK FFYDPLGSGR TFVDTVVSLT GFALVGGPAR QDAPKAIEAL KNLNVPYLVS LPLVFQTTEE WLDSELGVHP VQVALQVALP ELDGAMEPIV FAGRDSNTGK SHSLPDRIAS LCARAVNWAN LRKKRNAEKK LAVTVFSFPP DKGNVGTAAY LNVFGSIYRV LKNLQREGYD VGALPPSEED LIQSVLTQKE AKFNSTDLHI AYKMKVDEYQ KLCPYAEALE ENWGKPPGTL NTNGQELLVY GRQYGNVFIG VQPTFGYEGD PMRLLFSKSA SPHHGFAAYY TFLEKIFKAD AVLHFGTHGS LEFMPGKQVG MSGVCYPDSL IGTIPNLYYY AANNPSEATI AKRRSYANTI SYLTPPAENA GLYKGLKELK ELISSYQGMR ESGRAEQICA TIIETAKLCN LDRDVTLPDA DAKDLTMDMR DSVVGQVYRK LMEIESRLLP CGLHVVGCPP TAEEAVATLV NIAELDRPDN NPPIKGMPGI LARAIGRDIE SIYSGNNKGV LADVDQLQRI TEASRTCVRE FVKDRTGLNG RIGTNWITNL LKFTGFYVDP WVRGLQNGEF ASANREELIT LFNYLEFCLT QVVKDNELGA LVEALNGQYV EPGPGGDPIR NPNVLPTGKN IHALDPQSIP TQAALKSARL VVDRLLDRER DNNGGKYPET IALVLWGTDN IKTYGESLAQ VMMMVGVKPV ADALGRVNKL EVIPLEELGR PRVDVVVNCS GVFRDLFVNQ AVENSSWSDE SQLQEMYLKR KSYAFNSDRP GAGGEMQRDV FETAMKTVDV TFQNLDSSEI SLTDVSHYFD SDPTKLVASL RNDGRTPNAY IADTTTANAQ VRTLGETVRL DARTKLLNPK WYEGMLASGY EGVREIQKRM TNTMGWSATS GMVDNWVYDE ANSTFIEDAA MAERLMNTNP NSFRKLVATF LEANGRGYWD AKPEQLERLR QLYMDVEDKI EGVE

gun4
Mass: 24.36 kDa
Sequence:
MAMRVTVAAG KLDSVSLFGG DTASLMGGSQ TVEKKKSGKE AVMEVQLSST AGIDYTVLRD HLANGEFREA EDETRALLIK LAGPEAVKRN WVYFTEVKNI SVTDFQTLDN LWKASSNNKF GYSVQKEIWV QNQKRWPKFF KQIDWTQGEN NNYRKWPMEF IYSMDAPRGH LPLTNALRGT QLFQAIMEHP AFEKSSTAKT LDQKAAEAAG RTQSLF

References

[1] Adhikari ND, Froehlich JE, Strand DD, Buck SM, Kramer DM, Larkin RM. gUN4-Porphyrin Complexes Bind the chlH/gUN5 Subunit of Mg-Chelatase and Promote Chlorophyll Biosynthesis in Arabidopsis. Plant Cell. 2013; 23: 1449-1467.

[2] Tabrizi S, Sawicki A, Zhou S, Luo M, Willows R. GUN4-Protoporphyrin IX Is a Singlet Oxygen Generator with Consequences for Plastid Retrograde Signaling. Journal of Biological Chemistry. 2016;291(17): 8978-8984.