Team:Macquarie Australia/Project

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General background information on our project can be found Here.
A seperate 'Results & Characterization' section has been created to show and highlight our successful lab accomplishments, shown Here.

Overall project

This projects page aims to provide a general information of our project, without specifically following the lab notes which can be found Here
Current research into the elucidation of the chlorophyll biosynthetic pathway indicates that thirteen genes are necessary for successful chlorophyll production via several intermediates. The iGEM team at Macquarie University aims to synthetically create Biobrick versions of each of the genes responsible, with an end goal of their expression as a biosynthetic system in E. coli. This research will allow for strides forward in multiple disciplines.

Construction of this pathway will confirm or invalidate the current model for chlorophyll biosynthesis. It will also allow for exploration of the effectiveness of a synthetically produced photosystem II. Theory shows that electrons stripped from water by photosystem II could be passed on to an electron receiver or used to produced hydrogen fuel. Either of these methods will potentially allow for production of environmentally friendly energy.

Aims

Creation of Biobricks for the thirteen genes potentially responsible for chlorophyll biosynthesis
Construction of three promoted operons based on function
Production of biosynthetic pathway and qualification assays of protein function

Chlorophyll Biosynthesis Gene Pathway

The genes detailed below are necessary to construct our proposed chlorophyll synthesis pathway, within E. coli. In the figure below, each gene is represented by blue and each chlorophyll precursor is coloured according to their visual colour shown on expression. Each gene sequence has been modified for codon optimization, whilst maintaining protein integrity.



Chll1 - Magnesium chelatase subunit I
Forms an ATP dependent hexameric ring complex and a complex with the ChlD subunit (probably a double hexameric ring complex) before acting on the protoporphyrin which is bound to the ChlH protein to insert magnesium [PMID: 11469861]. Transcript is light regulated and may be diurnal and/or circadian [PMID: 16228385]; predicted chloroplast targeting sequence amino acids 1-54 by ChloroP.

Chll2 - Magnesium chelatase subunit I
forms an ATP dependent hexameric ring complex and a complex with the ChlD subunit (probably a double hexameric ring complex) before acting on the protoporphyrin which is bound to the ChlH protein to insert magnesium [PMID: 11469861]; may have similar function to Arabidopsis CHLI2 gene [PMID: 11842180]; chloroplast targeting signal peptide predicted 1-37 by ChloroP.

ChlD - Magnesium chelatase subunit D
Forms an ATP dependent complex with the ChlI subunit (probably a double hexameric ring complex) before acting on the protoporphyrin which is bound to the ChlH protein to insert magnesium [PMID: 11469861]. Predicted chloroplast targeting sequence amino acids 1-62 by ChloroP.

ChlH - Magnesium chelatase subunit H
chloroplast precursor; Chlamydomonas mutants with defects in this protein are chl1 and brs-1 and result in a brown phenotype [PMID: 11713666; PMID: 4436384]. Orthologous to the bacterial protein BchH [PMID: 9359397]; binds protoporphyrin and is acted upon by the ChlI:ChlD complex for magnesium insertion [PMID: 11469861]; interacts with GUN4 and may be involved in chloroplast signalling: Gene is also known as GUN5 in Arabidopsis thaliana [PMID: 11172074; 12574634]; transcript is light regulated and may be diurnal and/or circadian regulated [PMID: 16228385].

Gun4 - Tetrapyrrole-binding protein
In Arabidopsis, GUN4 (Genomes uncoupled 4) is required for the functioning of the plastid mediated repression of nuclear transcription that is involved in controlling the levels of magnesium- protoporphyrin IX. GUN4 binds the product and substrate of Mg-chelatase, an enzyme that produces Mg-Proto, and activates Mg-chelatase. GUN4 is thought to participates in plastid-to-nucleus signaling by regulating magnesium-protoporphyrin IX synthesis or trafficking.

ChlM - Mg protoporphyrin IX S-adenosyl methionine O-methyl transferase
Magnesium-protoporphyrin O-methyltransferase (chlM) [PMID: 12828371; PMID: 12489983; PMID: 4436384]; ChloroP 1.1 predicts cp location. ChlM is an important homologous enzyme involved in plastid-nucleus communication of plants. It is crucial for the methylation of magnesium protoporphyrin IX which is assembled by enzyme called “ChlM - Mg protoporphyrin IX S-adenosyl methionine O-methyl transferase”.

CTH1 - Copper target 1 protein
functional variant produced under copper and/or oxygen sufficient conditions [GI:15650866; PMID: 11910013; PMID: 14673103]; CTH1; Mg-protoporphyrin IX monomethyl ester (oxidative) cyclase, aerobic oxidative cyclase; orthologous to Rubrivaxgelatinosus aerobic oxidative cyclase [PMID: 11790744; PMID: 14617630]; predicted chloroplast transit peptide 1-35; Orthologous to CRD1; CHL27B [PMID: 15849308].

Plastocyanin - Chloroplast precursor
pre-apoplastocyanin, PETE [PMID: 2165059; PMID: 8940133]; structure of plastocyanin PDB: 2PLT; mutant = ac208 [PMID: 8463310] Plastocyanin contains copper and is a chloroplast precursor protein. It is taken up after post translation and placed on its functional site where it is involved in electron transfer between cytochrome f of the cytochrome b6f complex from photosystem II and P700+ from photosystem I.

POR - Light-dependent protochlorophyllidereductase
Light-dependent protochlorophyllidereductase, chloroplast precursor; Converts protochlorophyllide to chlorophyllide using NADPH and light as the reductant; Chlamydomonas mutant known as pc-1 has a two-nucleotide deletion within the fourth and fifth codons of this gene giving rise to a premature termination [PMID: 8616232; identical to U36752]

DVR1 - 3,8-divinyl protochlorophyllidea 8-vinyl reductase
Predicted chloroplast transit peptide 1-58; [PMID: 15695432; PMID: 15849308] It encodes for 3,8-divinyll Pchlide a 8-vinyl reductase that has important function in reduction of 8-vinyl group to the ethyl group on tetrapyrrole using NADPH as substrate. In addition to that, it is also responsible in conversion of divinyl protochlorophyllide a or divinylchlorophyllide to monovinyl protochlorophyllide a or monovinyl chlorophyllide via reduction of vinyl group.

ChlG - Chlorophyllsynthetase
Catalyses the esterification of chlorophyllide with phytyl-pyrophosphate to make chlorophyll A nuclear encoded gene which encodes chloroplast transit sequences for translocation of enzymes into the chloroplast using specific substrates. E.g. Phytyl-pyrophosphate and geranylgeranyl-pyrophosphate are substrates used by Avena sativa chlorophyll synthase.

ChlP - Geranylgeranyl reductase
Reduces the geranylgeranyl group to the phytyl group in the side chain of chlorophyll. Plant geranylgeranylhydrogenase (CHL P) reduces free geranylgeranyldiphosphate to phytildiphosphate, which provides the side chain to chlorophylls, tocopherols, and plastoquinones.

Methods and workflow

A quick summary of how we planned to approach the introduction of chlorophyll biosynthesis into E. coli

Design

We designed 10 genes necessary for chlorophyllide biosynthesis and another 2 genes for chlorophyll biosynthesis, totaling 12 genes. These genes were also codon optimised for expression within E. coli.

Assembly

Using Gibson Assembly we can reassemble our genes insert them into the plasmid backbone. This removes the need for ligations and restriction digests. Allowing the production of complete BioBricks without the need for extra steps to get the gene into the destination plasmid.

Transformation

By transforming in E. coli we can determine if the gene is functional as well as purify the plasmid. By transforming in top10 strain E. coli we can overproduce the proteins and then characterise the BioBricks produced.

Sequence

It is imperative that the plasmids produced from the Gibson Assembly be sequenced to determine if there have been any nucleotide changes between the planned sequences and those synthesised. Therefore sequencing data needs to be gathered before any ligations are performed to ensure the correct construction of our gene pathway. This will also demonstrate that the protein sequence has not changed and the protein should therefore be functional.

BioBrick Assembly

Following digestion of the BioBricks produced with the appropriate enzyme and ligation it is possible to produce the plasmids required for chlorophyll biosynthesis. This protocol can be seen below,

Transformation & Characterizations

After ligating BioBricks to assemble our gene pathway we will be able to show the usefulness of Gibson Assembly in synthetic biology. This will provide a means to characterise the two biobricks simultaneously.

Highlighted results

Shown here are some of our most important and successful results, summarized.

Gene Sequencing Results - All of our genes have been shown to be ligated correctly from gBlocks, all our sequencing results have comeback with a identity match of 99% or higher









[Part 2-The Experiments-Part 3-Results]

Future/Significance of project

Back to the Future IVXC