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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 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.

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 150kDa chlH protein is highly expressed and visible on SDS-PAGE and confirmed by proteomic analysis. This should sufficient to produce Mg-Protoporphyrin IX.

2. Mg-protoporphyrin is not produced in DH5-alpha E.coli containing this plasmid when induced with IPTG. However, Mg-protoporphyrin IX is produced in a hemH mutant background of E.coli containing this plasmid when induced with IPTG. (here shauna, add the link to proof of concept). This ΔhemH<i> strain demonstrates high levels of protoporphyrin IX production within the cell.

3. The Mg-protoporphyrin IX produced in this strain demonstrates that metabolic engineering of E. coli is required to generate sufficient protoporphyrin IX substrate for the assembled magnesium chelatase complex to successfully make Mg-protoporphyrin IX.

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

1. The digests of Mg-chelatase plasmid reveal the correct band sizes of the insert (approx 11 kbp). ChlH protein (approx. 150kDa) is also highly expressed and visible on SDS-PAGE.</font></div>

</center> 2. Mass spectrometry confirms the presence of magnesium chelatase subunits</div>
<center>

<img src=" ", alt="important 1st " height="200%" width="400"> <figcaption>

Fig 1. 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 <i>ChlH</i> protein. The MW of other proteins of interest include <i>ChlI1</i> (40 kDa), <i>ChlD</i> (63 kDa), Gun4 (24 kDa), <i>ChlI2</i> (40 kDa) and CTH1 (43 kDa).</center></figcaption></div>

<img src="", alt="important 1st " height="200%" width="400"> <figcaption>

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></figcaption></div>

<img src=" ", alt="important 1st " height="200%" width="400"> <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>

<img src="", alt="hemH Mutants" height="250px" width="400px"> <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>

</center> 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.</div>

<center> </center>

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

<center>

<img src="", alt="hemH Mutants" height="200%" width="450"> <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>

<img src="", alt="hemH mutants 2" height="200%" width="400"> <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>

Protein information

<i>chlI1</i>
Mass: 39.96kDa
Sequence:
MAATEVKAAEGRTEKELGQARPIFPFTAIVGQDEMKLALILNVIDPKIGGVMIMGDRGTGKSTTIRALADLLPEMQVVANDPFNSDPTDPELMSEEVRNR VKAGEQLPVSSKKIPMVDLPLGATEDRVCGTIDIEKALTEGVKAFEPGLLAKANRGILYVDEVNLLDDHLVDVLLDSAASGWNTVEREGISISHPARFIL VGSGNPEEGELRPQLLDRFGMHAQIGTVKDPRLRVQIVSQRSTFDENPAAFRKDYEAGQMALTQRIVDARKLLKQGEVNYDFRVKISQICSDLNVDGIRG DIVTNRAAKALAAFEGRTEVTPEDIYRVIPLCLRHRLRKDPLAEIDDGDRVREIFKQVFGME

<i>chlD</i>
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

<i>gun4</i>
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

<i>chlI2</i>
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

<i>cTH1</i>
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

<i>chlH</i>
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. <i>gUN4</i>-Porphyrin Complexes Bind the <i>chlH/gUN5</i> 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.