Device

Part:BBa_K1998000

Designed by: Shauna Winchester   Group: iGEM16_Macquarie_Australia   (2016-10-12)
Revision as of 01:45, 21 October 2016 by Rwillows (Talk | contribs) (→‎Characterisation and Verification)


Mg-Chelatase Plasmid

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 4753
    Illegal NotI site found at 2666
    Illegal NotI site found at 4213
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 439
    Illegal BglII site found at 1095
    Illegal BglII site found at 1290
    Illegal BglII site found at 3371
    Illegal BglII site found at 3898
    Illegal BglII site found at 5755
    Illegal BglII site found at 5863
    Illegal BamHI site found at 487
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1946
    Illegal NgoMIV site found at 2765
    Illegal NgoMIV site found at 3307
    Illegal NgoMIV site found at 7968
    Illegal AgeI site found at 6853
    Illegal AgeI site found at 6883
    Illegal AgeI site found at 7777
    Illegal AgeI site found at 7945
    Illegal AgeI site found at 9463
    Illegal AgeI site found at 9517
    Illegal AgeI site found at 9736
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 5553
    Illegal BsaI.rc site found at 5709
    Illegal BsaI.rc site found at 6330
    Illegal BsaI.rc site found at 9862
    Illegal SapI.rc site found at 7219
    Illegal SapI.rc site found at 9792


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 six biobricks previously registered by a past Macquarie iGEM team. These genes form the first part of the chlorophyll biosynthesis pathway. The six genes are in the order of chlI1, chlD, gun4, chlI2, cTH1, followed by chlH. At the beginning of each gene, a ribosome binding site can be found. plac is also located at the front of chlH. Another plac is at the beginning of chlI1. The genes encode for Magnesium chelatase subunit H (chlH), Magnesium chelatase subunit I (chli1/chli2), Magnesium chelatase subunit D (chlD), Genomes uncoupled 4 (gUN4) and Copper target 1 protein (cth1). These genes are involved in converting protoporphyrin IX to divinyl protochlorophyllide in the presence of NADPH and O2.

Biology & Literature

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 an ATP dependent hexameric ring complex and a complex with the chlD subunit. This complex then acts on protoporphyrin. The gUN4 gene encodes genomes uncoupled 4 protein. This is a tetrapyrrole-binding protein which controls the production of Mg-protoporhyrin IX. The cTH1 gene encodes for a copper target protein. It forms when oxygen and copper are present catalysing Mg-protoporphyrin IX mono methyl, and subsequently converts to divinyl protochlorophyllide when NADPH and O2 are present. The last gene is chlH. This gene encodes for the magnesium shelters subunit H. This acts as a chloroplast precursor, catalysing the first step in the chlorophyl biosynthesis pathway by insertion of an Mg2+ ion into protoporphyrin IX to generate Mg-protoporphyrin IX.

The chlH and gUN4 proteins bind to protoporphyrin IX and form an activated substrate complex which behaves as a substrate for the motor complex (ChlID complex) to insert magnesium into the bound Protoporphyrin IX upon ATP hydrolysis. The assembly of this complex requires milliMolar concentrations of Mg2+ and ATP to assemble. While the substrate complex also requires microMolar concentrations of Protoporphyrin IX and chlH for optimal activity.

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Assembly and Design

Each individual biobrick was assembled in the following order using 3A assembly: pLac-chlI1-chlD-gun4-chlI2-cTH1-pLac-chlH [BBa_R0010], [BBa_K1326008], [BBa_K1080011], [BBa_K1080005] and [BBa_K1640019]. Five of these genes code for different subunits of the enzyme Magnesium chelatase, with the cTH1 gene encoding a component of a later enzyme in the pathway, the oxidative cyclase.

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

Characterisation and Verification

The following information shows the characterisation process of this plasmid as well as it's verification.

1. The digests of Mg-chelatase plasmid reveal the correct band sizes of the insert (approx 11 kip) as shown in Fig 1. ChlH protein (approx. 150kDa) is also highly expressed and visible on SDS-PAGE as shown in Fig 2.

important 1st

Fig 1.Digests of Plasmids

2. Mass spectrometry confirms the presence of magnesium chelatase subunits.

important 1st


Fig 2. Protein expression of the Mg-chelatase plasmid induced with IPTG. Lane 2 is the uninduced culture. Highly expressed band at approximately 144 kDa represents the ChlH protein. The MW of other proteins of interest include ChlI1 (40 kDa), ChlD (63 kDa), Gun4 (24 kDa), ChlI2 (40 kDa) and CTH1 (43 kDa).


<center> important 1st

<figcaption>
Fig 3. All bands the size of the proteins of interest expressed from the induced magnesium chelatase plasmid were extracted from the SDS PAGE. MALDI TOF analysis of these proteins revealed that ChlH and ChlI2 were expressed successfully according to GPM.</center></figcaption></div>


<center> hemH Mutants

<figcaption>
Fig 4. We observe the increase in MgPPIX concentration at day 3 in the figure above. According to graphs produced on the <a class="regularHyperlink" href="http://2016.igem.org/Team:Macquarie_Australia/Model">modelling page</a>, that is when PPIX is starting to be generated. We therefore conclude that we only get accumulation of MgPPIX when PPIX increases within the cell and when we induce with IPTG.</center></figcaption></div>



3. Mg-protoporphyrin is not produced in DH5-alpha E.coli containing this plasmid even when induced with IPTG. However, the deltahemH strain created via CRISPR accumulates protoporphyrin IX within the cell.

4. Transformed Mg chelatase plasmid into ΔhemH mutants and it produced an increasing amount of Mg-PPIX demonstrating that the metabolic engineering of E. coli is required to generate sufficient PPIX substrate for the assembled magnesium chelatase complex to function. hemH Mutants

<figcaption>
Fig 5. Fluorescence excitation and emission spectra demonstrates the production of Mg-PPIX upon the addition of Mg-chelatase plasmid to ΔhemeH mutants. Mg-PPIX has an emission and excitation spectra of 418nm and 592nm respectively.</center></figcaption></div>

hemH mutants 2

<figcaption>
Fig 6. Emission and excitation spectra of the in vitro assay of Mg-chelatase from the Mg-P plasmid. Zero time control is the initial emission/excitation spectra when Mg-chelatase is added to 10mM PPIX, 10mM ATP and 15mM MgCl2. After incubating the assay under constant reagents as the control at room temperature, Mg-PPIX production increases. When additional 50mM MgCl2 is added to the assay in comparison to the control, Mg-PPIX production increases further. However, upon addition of 50mM MgCl2 including 20nM of ChlID complex, the production of Mg-PPIX remains the same. Since adding additional ChlID enzyme to the last assay does not change Mg-PPIX production, the ChlID complex is not limiting.</center></figcaption></div>

Further information can be found here: http://2016.igem.org/Team:Macquarie_Australia/Proof

Protein information

chlI1
Mass: 39.96kDa
Sequence:
MAATEVKAAEGRTEKELGQARPIFPFTAIVGQDEMKLALILNVIDPKIGGVMIMGDRGTGKSTTIRALADLLPEMQVVANDPFNSDPTDPELMSEEVRNR VKAGEQLPVSSKKIPMVDLPLGATEDRVCGTIDIEKALTEGVKAFEPGLLAKANRGILYVDEVNLLDDHLVDVLLDSAASGWNTVEREGISISHPARFIL VGSGNPEEGELRPQLLDRFGMHAQIGTVKDPRLRVQIVSQRSTFDENPAAFRKDYEAGQMALTQRIVDARKLLKQGEVNYDFRVKISQICSDLNVDGIRG DIVTNRAAKALAAFEGRTEVTPEDIYRVIPLCLRHRLRKDPLAEIDDGDRVREIFKQVFGME

chlD
Mass: 63.84kDa
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

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

chlI2
Mass: 39.56kDa
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

cTH1
Mass: 43.88kDa
Sequence:
MVAATAAPQE VEGFKVMRDG IKVASDETLL TPRFYTTDFD EMERLFSLEL NKNMDMEEFE AMLNEFKLDY NQRHFVRNET FKEAAEKIQG PTRKIFIEFL ERSCTAEFSG FLLYKELGRR LKATNPVVAE IFTLMSRDEA RHAGFLNKAM SDFNLALDLG FLTKNRKYTF FKPKFIFYAT YLSEKIGYWR YISIYRHLQR NPDNQLYPLF EYFENWCQDE NRHGDFFTAV LKARPEMVND WAAKLWSRFF CLSVYITMYL NDHQRDAFYS SLGLNTTQFN QHVIIETNKS TERIFPAVPD VENPEFFRRM DLLVKYNAQL VNIGSMNLPS PIKAIMKAPI LERMVAEVFQ VFIMTPKESG SYDLDANKTA LVY

chlH
Mass:144.14
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

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.