Difference between revisions of "Part:BBa K2664007"

Revision as of 10:55, 17 October 2018

Protochlorophyllide to chlorophyll-a plasmid

Conversion of protochlorophyllide to chlorophyll a

Sequence and Features

Overview

This composite part corresponds to the four final genes of the complete chlorophyll biosynthesis pathway [BBa_K2664008] and it is composed of POR,ChlP, DVR1 and ChlG. These genes code for the Light-dependent protochlorophyllide reductase (POR), 3,8-divinyl Geranylgeranyl reductase (ChlP), protochlorophyllide-a 8-vinyl reductase (DVR1) and Chlorophyll synthetase (ChlG).
Being the last parts in the pathway, they are responsible for conversion of divinyl protochlorophyllide to chlorophyll a. The divinyl protochlorophyllide is the precursor of chlorophyll a and is generated from Mg-protoporphyrin IX, after the interference of other enzymes used in the full pathway.

Biology & Literature

The POR gene is a light dependent protochlorophyllide reductase. Its role is to convert protochlorophyllide to chlorophyllide using NADPH and light as the reductants. Formation of the POR protein in plants is crucial, as without it the chloroplasts do not function [1,2]. The protochlorophyllide is reduced to chlorophyllide through light excitation, functioning as a catalyst. The excitation of the POR molecule and its conversion to chlorophyllide causes the membranes to turn from an inactive state into the active chloroplast [3]. Through the transformation into the active state, the chloroplasts produce that be used by the plant.

The ChlP gene encodes for an enzyme, the geranylgeranyl reductase, which catalyses the addition of the geranylgeranyl pyrophosphate chain into the chlorophyllide molecule. The reaction occurs through hydrogenation of geranylgeranyl diphosphate (GGPP), that reduces the molecule’s (GGPP) double bonds [4].

The DVR1 gene encodes for 3,8-divinyl protochlorophyllide 8-vinyl reductase, which reduces divinly protochlorophyllide a to monovinyl protochlorophyllide a [5]. This is where the POR gene comes in in the pathway, catalysing the reduction of protochlorophyllide to chlorophyllide.

The final gene in this part and the complete pathway is the ChlG gene, which encodes the enzyme chlorophyll synthetase. As the name implies, it is the final step in chlorophyll synthesis, which is achieved after chlorophyll synthetase catalyses the esterification of chlorophyllide with GGPP.

Assembly and Design

Each individual biobrick was assembled in the following order using standard assembly:
trc-POR-ChlP-DVR1-ChlG.
The basic biobricks corresponding to the genes were:
[BBa_K2664001], [BBa_K1080008], [BBa_K1080012] and [BBa_K1080009], respectively.

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

Part Verification

Fig 1. Gel electrophoresis (1% agarose) provides evidence of successful assembly of Mg-PPIX to Chlorophyll a plasmid (6.3 kbp) (Lane 5). Plasmid was assembled via 3A assembly and double digested to reveal the biobrick backbone (2000 bp) and the correct insert size.

Protein information

POR
Mass: 41.87kDa
Sequence:
MVVCAATATAPSPSLADKFKPNAIARVPATQQKQTAIITGASSGLGLNAAKALAATGEWHVVMACRDFLKAEQAAKKVGMPAGSYSILHLDLSSLESVRQFVQNFKASGR RLDALVCNAAVYLPTAKEPRFTADGFELSVGTNHLGHFLLTNLLLDDLKNAPNKQPRCIIVGSITGNTNTLAGNVPPKANLGDLSGLAAGVPAANPMMDGQEFNGAKAYK DSKVACMMTV RQMHQRFHDATGITFASLYPGCIAETGLFREHVPLFKTLFPPFQKYITKGYVSEEEAGRRLAAVISDPKLNKSGAYWSWSSTTGSFDNK

ChlP
Mass: 47kDa
Sequence:
MVIGGGPSGACAAETLAKGGVETFLLERKLDNCKPCGGAIPLCMVEEFDLPMEIIDRRVTKMKMISPSNREVDVGKTLSETEWIGMCRREVFDDYLRNRAQKLGANIVNGL FMRSEQQSAEGPFTIHYNSYEDGSKMGKPATLEVDMIIGADGANSRIAKEIDAGEYDYAIAFQERIRIPDDKMKYYENLAEMYVGDDVSPDFYGWVFPKYDHVAVGTGTVVN KTAIKQYQQATRDRSKVKTEGGKIIRVEAHPIPEHPRPRRCKGRVALVGDAAGYVTKCSGEGIYFAAKSGRMAAEAIVEGSANGTKMCGEDAIRVYLDKWDRKYWTTYKVLD ILQKVFYRSNPAREAFVELCEDSYVQKMTFDSYLYKTVVPGNPLDDVKLLVRTVSSILRSNALRSVNSKSVNVSFGSKANEERVM AA

DVR1

Mass: 37kDa
Sequence:
MAMAASRQAVRVAAAVDADYRKREPKDVRVLVVGPTGYIGKFVVKELVSRGYNVVAFARENAGIKGKMGREDIVKEFHGAEVRFGSVLDPASLRDVAFKDPVDVVVSCLA SRTGGKKDSWLIDYTATKNSLDVARASGAKHFVLLSAICVQKPLLEFQKAKLQFESDLQAAGDITYSIVRPTAFFKSIAGQIDIVKKGNPYVMFGDGNLAACKPISEADLASF IADCVTEQNKVNKVLPIGGPSKAFTAKQQADLLFNITGLPPKYFPVPVALMDGMIGLFDSLAKLFPQLEDSAEFARIGKYYATESMLVYDEARGVYRKTKRLVTARTRWKTS SLVQ

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

References

[1] Fujita Y. Protochlorophyllide reduction: a key step in the greening of plants. Plant and cell physiology. 1996 Jun 1;37(4):411-21.

[2] Darrah PM, Kay SA, Teakle GR, Griffiths WT. Cloning and sequencing of protochlorophyllide reductase. Biochemical Journal. 1990 Feb 1;265(3):789-98.

[3] Griffiths WT. Characterization of the terminal stages of chlorophyll (ide) synthesis in etioplast membrane preparations. Biochemical Journal. 1975 Dec 1;152(3):623-55.

[4] Shpilyov AV, Zinchenko VV, Shestakov SV, Grimm B, Lokstein H. Inactivation of the geranylgeranyl reductase (ChlP) gene in the cyanobacterium Synechocystis sp. PCC 6803. Biochimica et Biophysica Acta (BBA)-Bioenergetics. 2005 Feb 17;1706(3):195-203.

[5] Davies K, editor. Annual plant reviews, plant pigments and their manipulation. John Wiley & Sons; 2009 Feb 12.