Difference between revisions of "Part:BBa K2664008"

(→‎Biology & Literature)
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===Overview===
 
===Overview===
  
−
This series of genes derived from the algae <i>Chlamydomonas reinhardtii</i> have been brought together to express the complete chlorophyll biosynthesis pathway in <i>Escherichia coli</i>. In 2018 the Team Macquarie Australia has made rearrangements in existing parts of the pathway, introduced new genes and swapped plac with the trc promoter. The parts have been assembled to allow for gradual transition from Protoporphyrin IX to Chlorophyll and we are confident that our improved plasmid will be more efficient at producing chlorophyll, than its previous versions.
+
This series of genes derived from the algae <i>Chlamydomonas reinhardtii</i> have been brought together to express the complete chlorophyll biosynthesis pathway in <i>Escherichia coli</i>. In 2018 Team Macquarie Australia has made rearrangements in existing parts of the pathway, introduced new genes and swapped lac with the trc promoter. The parts have been assembled to allow for gradual transition from Protoporphyrin IX to Chlorophyll and we are confident that our improved plasmid will be more efficient at producing chlorophyll, than its previous versions.
  
 
===Biology & Literature===
 
===Biology & Literature===
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3. Final reaction from Protochlorophyllide to Chlorophyll-a<br><br>
 
3. Final reaction from Protochlorophyllide to Chlorophyll-a<br><br>
  
−
The first reaction involves the genes <i>ChlI1, ChlI2, ChlD, ChlH</i> and <i>GUN4</i>. To begin with, <i>ChlI1</i> and <i>ChlI2</i> form the initial Mg-chelatase complex (Magnesium chelatase subunit I). This complex catalyses the insertion of a magnesium ion into protoporphyrin IX to yield Mg-protoporphyrin IX. This happens through the formation of two different complexes, an ATP dependent hexameric ring complex and a complex with the chlD subunit.
+
The first reaction involves the genes <i>ChlI1, ChlI2, ChlD, ChlH</i> and <i>GUN4</i>. To begin with, <i>ChlI1</i> and <i>ChlI2</i> form the initial Mg-chelatase complex (Magnesium chelatase subunit I). This complex catalyses the insertion of a magnesium ion into protoporphyrin IX to yield Mg-protoporphyrin IX. This happens through the formation of two different complexes, an ATP dependent hexameric ring complex and a complex with the chlD subunit.<br>
 +
 
  
 
===Assembly and Design===
 
===Assembly and Design===
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===References===
 
===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.
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Revision as of 23:49, 17 October 2018


Complete chlorophyll expression pathway

Complete chlorophyll expression pathway

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1720
    Illegal NotI site found at 3751
    Illegal NotI site found at 9504
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1
    Illegal BglII site found at 304
    Illegal BglII site found at 960
    Illegal BglII site found at 1155
    Illegal BglII site found at 4456
    Illegal BglII site found at 4580
    Illegal BglII site found at 6580
    Illegal BamHI site found at 352
    Illegal BamHI site found at 10997
    Illegal BamHI site found at 16476
    Illegal BamHI site found at 17457
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 3031
    Illegal NgoMIV site found at 3850
    Illegal NgoMIV site found at 4392
    Illegal NgoMIV site found at 5138
    Illegal NgoMIV site found at 6804
    Illegal NgoMIV site found at 12155
    Illegal AgeI site found at 5784
    Illegal AgeI site found at 7302
    Illegal AgeI site found at 7356
    Illegal AgeI site found at 7575
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 11159
    Illegal BsaI.rc site found at 6906
    Illegal BsaI.rc site found at 7701
    Illegal BsaI.rc site found at 9832
    Illegal BsaI.rc site found at 9988
    Illegal BsaI.rc site found at 10609
    Illegal BsaI.rc site found at 14945
    Illegal SapI.rc site found at 7631

Overview

This series of genes derived from the algae Chlamydomonas reinhardtii have been brought together to express the complete chlorophyll biosynthesis pathway in Escherichia coli. In 2018 Team Macquarie Australia has made rearrangements in existing parts of the pathway, introduced new genes and swapped lac with the trc promoter. The parts have been assembled to allow for gradual transition from Protoporphyrin IX to Chlorophyll and we are confident that our improved plasmid will be more efficient at producing chlorophyll, than its previous versions.

Biology & Literature

The chlorophyll biosynthesis pathway could be divided in three major reactions:
1. Magnesium chelation to protoporphyrin IX give rise to Mg-Protoporphyrin IX
2. Conversion of Mg-Protoporphyrin IX to Protochlorophyllide
3. Final reaction from Protochlorophyllide to Chlorophyll-a

The first reaction involves the genes ChlI1, ChlI2, ChlD, ChlH and GUN4. To begin with, ChlI1 and ChlI2 form the initial Mg-chelatase complex (Magnesium chelatase subunit I). This complex catalyses the insertion of a magnesium ion into protoporphyrin IX to yield Mg-protoporphyrin IX. This happens through the formation of two different complexes, an ATP dependent hexameric ring complex and a complex with the chlD subunit.


Assembly and Design

HydrogenProduction

Figure 1. Graphic representation of the designing process for assembling the complete chlorophyll biosynthesis pathway in E. coli. The process was completed using standard assembly.

Part Verification

Figure 2. Agarose gel electrophoresis (0.5% agarose) with GelRed (2 μl/100 ml) showing single (E) and double (E+P) digests of the complete plasmid for Chlorophyll a biosynthesis pathway (18352bp). The 1kb gene ruler plus was loaded in Lane 1 for size reference.


Protein Information

ChlI1
Mass: 39.95 kDa
Sequence:
MAATEVKAAE GRTEKELGQA RPIFPFTAIV GQDEMKLALI LNVIDPKIGG VMIMGDRGTG KSTTIRALAD LLPEMQVVAN DPFNSDPTDP ELMSEEVRNR VKAGEQLPVS SKKIPMVDLP LGATEDRVCG TIDIEKALTE GVKAFEPGLL AKANRGILYV DEVNLLDDHL VDVLLDSAAS GWNTVEREGI SISHPARFIL VGSGNPEEGE LRPQLLDRFG MHAQIGTVKD PRLRVQIVSQ RSTFDENPAA FRKDYEAGQM ALTQRIVDAR KLLKQGEVNY DFRVKISQIC SDLNVDGIRG DIVTNRAAKA LAAFEGRTEV TPEDIYRVIP LCLRHRLRKD PLAEIDDGDR VREIFKQVFG ME

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

CTH1
Mass: 43.87 kDa
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

YCF54
Mass: 17.07 kDa
Sequence:
MAPAAASADK ATAAEYYALV CNAEWFFMDP QNESVAEQLR EKVRFFKEQN KERDFFIVPN PKWLDAKFPE QAKQVKRPCV ALVSTDKMWI TFMKLRLDRV LKIDLKSMPA SEVLAAGEAL PDFKPDGKWT APYARYTPGW WNVFLPNH

ChlM
Mass: 30.44 kDa
Sequence:
MASEIAQTAD VGSLTFAVGG VGAVVGLGAL LVATDHQKRR SEQMKSFDGD EKEAVKDYFN TAGFERWRKI YGETDEVNKV QLDIRTGHAQ TVDKVLRWVD EEGSVQGITV ADCGCGTGSL AIQLALRGAA VSASDISAAM ASEAEQRYQQ AVAAGQGKAP KVAPKFEALD LESVKGKYDT VTCLDVMIHY PQDKVDAMIT HLAGLSDRRL IISFAPKTLS YSILKRIGEL FPGPSKATRA YLHREEDVEA ALKRAGFKVT KREMTATSFY FSRLLEAIRE

Ferredoxin
Mass: 13.0 kDa
Sequence:
MAMRSTFAARVGAKPAVRGARPASRMSCMAYKVTLKTPSGDKTIECPADTYILDAAEEAGLDLPYSCRAGACSSCAGKVAAGTVDQSDQSFLDDAQMGNGFV LTCVAYPTSDCTIQTHQEEALY

Ferredoxin NADP+ reductase (FNR)
Mass: 38.27 kDa
Sequence:
MQTVRAPAASGVATRVAGRRMCRPVAATKASTAVTTDMSKRTVPTKLEEGEMPLNTYSNKAPFKAKVRSVEKITGPKATGETCHIIIETEGKIPFWEGQSYGVIPP GTKINSKGKEVPHGTRLYSIASSRYGDDFDGQTASLCVRRAVYVDPETGKEDPAKKGLCSNFLCDATPGTEISMTGPTGKVLLLPADANAPLICVATGTGIAPFRS FWRRCFIENVPSYKFTGLFWLFMGVANSDAKLYDEELQAIAKAYPGQFRLDYALSREQNNRKGGKMYIQDKVEEYADEIFDLLDNGAHMYFCGLKGMMPGIQD MLERVAKEKGLNYEEWVEGLKHKNQWHVEVY

POR
Mass: 41.87 kDa
Sequence:
MVVCAATATAPSPSLADKFKPNAIARVPATQQKQTAIITGASSGLGLNAAKALAATGEWHVVMACRDFLKAEQAAKKVGMPAGSYSILHLDLSSLESVRQFVQNFKASGR RLDALVCNAAVYLPTAKEPRFTADGFELSVGTNHLGHFLLTNLLLDDLKNAPNKQPRCIIVGSITGNTNTLAGNVPPKANLGDLSGLAAGVPAANPMMDGQEFNGAKAYK DSKVACMMTV RQMHQRFHDATGITFASLYPGCIAETGLFREHVPLFKTLFPPFQKYITKGYVSEEEAGRRLAAVISDPKLNKSGAYWSWSSTTGSFDNK

ChlP
Mass: 47 kDa
Sequence:
MVIGGGPSGACAAETLAKGGVETFLLERKLDNCKPCGGAIPLCMVEEFDLPMEIIDRRVTKMKMISPSNREVDVGKTLSETEWIGMCRREVFDDYLRNRAQKLGANIVNGL FMRSEQQSAEGPFTIHYNSYEDGSKMGKPATLEVDMIIGADGANSRIAKEIDAGEYDYAIAFQERIRIPDDKMKYYENLAEMYVGDDVSPDFYGWVFPKYDHVAVGTGTVVN KTAIKQYQQATRDRSKVKTEGGKIIRVEAHPIPEHPRPRRCKGRVALVGDAAGYVTKCSGEGIYFAAKSGRMAAEAIVEGSANGTKMCGEDAIRVYLDKWDRKYWTTYKVLD ILQKVFYRSNPAREAFVELCEDSYVQKMTFDSYLYKTVVPGNPLDDVKLLVRTVSSILRSNALRSVNSKSVNVSFGSKANEERVMAA

DVR1
Mass: 37 kDa
Sequence:
MAMAASRQAVRVAAAVDADYRKREPKDVRVLVVGPTGYIGKFVVKELVSRGYNVVAFARENAGIKGKMGREDIVKEFHGAEVRFGSVLDPASLRDVAFKDPVDVVVSCLA SRTGGKKDSWLIDYTATKNSLDVARASGAKHFVLLSAICVQKPLLEFQKAKLQFESDLQAAGDITYSIVRPTAFFKSIAGQIDIVKKGNPYVMFGDGNLAACKPISEADLASF IADCVTEQNKVNKVLPIGGPSKAFTAKQQADLLFNITGLPPKYFPVPVALMDGMIGLFDSLAKLFPQLEDSAEFARIGKYYATESMLVYDEARGVYRKTKRLVTARTRWKTS SLVQ

ChlG
Mass: 36.84 kDa
Sequence:
MNQQATEEKSDTNSAARQMLGMKGAALETDIWKIRVQLTKPVTWIPLIWGVACGAAASGHYQWNNPTQIAQLLTCMMMSGPFLTGYTQTINDWYDREIDAINEPYRPIPS GRISERDVIVQIWVLLLGGIGLAYTLDQWAGHTTPVMLQLTIFGSFISYIYSAPPLKLKQSGWAGNYALGSSYIALPWWAGQALFGTLTLDVMALTIAYSLAGLGIAIVNDFKSI EGDRQ MGLQSLPVAFGVDTAKWICVSTIDVTQLGVAAYLAWGLHEELYGAVLLALILPQIYFQYKYFLPDPIANDVKYQASAQPFLVFGLLTAGLACGHHVNAVAA AASAAGAL

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.