Team:Macquarie Australia/Project
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General
A seperate
Overall project
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
[Amy's Aim?]
Chlorophyll Biosynthesis Gene Pathway
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
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]
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]
ChlG - Chlorophyllsynthetase
Catalyses the esterification of chlorophyllide with phytyl-pyrophosphate to make chlorophyll
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
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
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]