Team:Macquarie Australia/Project

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<h1><center>Overall project</center></h1>
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<center><b><font size=5>[Page Under Construction]</font size></b></center>
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<center><h7> This projects page aims to provide a general information of our project, without specifically following the lab notes which can be found <td></td><td><span style="color:#8B0000"><font size = 3><a class="three" href='https://2013.igem.org/Team:Macquarie_Australia/Notebook'><b>Here</b></a></font size></h7></span>
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<span style="color:#8B0000"><font size = 3><a class="three" href='http: //2013.igem.org/Team:Macquarie_Australia/Project/background'><b>Background link</b></a></font size></span>
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General <td></td><td><span style="color:#8B0000"><font size = 3><a class="three" href='http: //2013.igem.org/Team:Macquarie_Australia/Project/background'><b>background</b></a></font size></span> information on our project can be found <td></td><td><span style="color:#8B0000"><font size = 3><a class="three" href='http: //2013.igem.org/Team:Macquarie_Australia/Project/background'><b>Here</b></a></font size></span>.
 
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A seperate
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<td></td><td><span style="color:#8B0000"><font size = 3><a class="three" href='https://2013.igem.org/Team:Macquarie_Australia/Results'><b>Results link</b></a></font size></span> </center>
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<td></td><td><span style="color:#8B0000"><font size = 3><a class="three" href='https://2013.igem.org/Team:Macquarie_Australia/Results'><b>'Results & Characterization'</b></a></font size></span> section has been created to show and highlight our successful lab accomplishments, shown
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<td></td><td><span style="color:#8B0000"><font size = 3><a class="three" href='https://2013.igem.org/Team:Macquarie_Australia/Results'><b>Here</b></a></font size></span>.
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<h1><center>Overall project</center></h1>
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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 <i>E. coli</i>. This research will allow for strides forward in multiple disciplines.
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<center><h7> This projects page aims to provide a general information of our project, without specifically following the lab notes which can be found <td></td><td><span style="color:#8B0000"><font size = 3><a class="three" href='https://2013.igem.org/Team:Macquarie_Australia/Notebook'><b>Here</b></a></font size></h7></span></center>
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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.
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<b><center><font size=5>Aims</font size></b></center>
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The iGEM team at Macquarie University are aiming to introduce the genes necessary for chlorophyll production into E.coli. We at Macquarie are confident that we can make scientific strides in the understanding and construction of a photosynthetic bacterium.<br><br>
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Currently we have 2 out of 12 biobricks assembled and sequenced in our system, with work continuing on the remainder.
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<font size=3>•</font><h7>Creation of Biobricks for the thirteen genes potentially responsible for chlorophyll biosynthesis.</h7>
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Production of chlorophyll in E.coli would be the first steps towards the construction of photosystem II, a fundamental aspect of organic energy production. A better understanding of photosystem II opens the door to the production of harnessing green energy.
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<font size=3>•</font><h7>Construction of three promoter operons based on function.</h7>
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<font size=3>•</font><h7>Production of a biosynthetic pathway and qualification assays of protein function</h7>
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If successful this would be the first successful production of chlorophyll within non-photosynthetic bacteria.
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<b><font size=4>Aims</font size></b>
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<center><img src = https://static.igem.org/mediawiki/2013/7/76/Summary_Pic2.png width = 750></center><br>
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<b>Figure -</b> Our construction of three operons to synthesise chlorophyll from protophoryphyrin iX in <i>E. coli</i>.
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<h1><center>Chlorophyll Biosynthesis Gene Pathway</center></h1>
<h1><center>Chlorophyll Biosynthesis Gene Pathway</center></h1>
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<center><h7> The synthetic recreation of the chlorophyll biosynthesis pathway within <i>E. coli</i> is our goal...Sequences of genes have been modified for expression within E.coli </h7></center>
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<center><h7><p> The genes detailed below are necessary to construct our proposed chlorophyll synthesis pathway, within <i>E. coli</i>. 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. </p></h7></center>
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<img src="https://static.igem.org/mediawiki/2013/a/ae/Chloropyllpathway4.jpg" alt="Smiley face" align="right">
<img src="https://static.igem.org/mediawiki/2013/a/ae/Chloropyllpathway4.jpg" alt="Smiley face" align="right">
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<font size = 5><span style="color:#333"><b>Protoporphyrin IX</b></font size> </span>
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<b><font size=3>Chll1</font size>
<b><font size=3>Chll1</font size>
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  - Magnesium chelatase subunit I</b>
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  - Magnesium Chelatase subunit I</b>
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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.  
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Catalyzes the insertion of magnesium ion into protoporphyrin IX to yield Mg-protoporphyrin IX.
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Forms an ATP dependent hexameric ring complex and a complex with the ChlD subunit. Transcript is light regulated and may be diurnal and/or circadian.
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<b><font size=3>Chll2</font size>
<b><font size=3>Chll2</font size>
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- Magnesium chelatase subunit I</b>
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- Magnesium Chelatase subunit I</b>
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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.  
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The second gene which catalyzes the insertion of magnesium ion into protoporphyrin IX to yield Mg-protoporphyrin IX. Forms an ATP dependent hexameric ring complex and a complex with the ChlD subunit.
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<b><font size=3>ChlD</font size>
<b><font size=3>ChlD</font size>
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- Magnesium chelatase subunit D</b>
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- Magnesium Chelatase subunit D</b>
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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.  
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Forms an ATP dependent complex with the ChlI subunits 1 & 2, before acting on the protoporphyrin which is bound to the ChlH protein to insert magnesium.
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<b><font size=3>ChlH</font size>
<b><font size=3>ChlH</font size>
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- Magnesium chelatase subunit H</b>
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- Magnesium Chelatase subunit H</b>
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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].  
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Involved in bacteriochlorophyll pigment biosynthesis; introduces a magnesium ion into protoporphyrin IX to yield Mg-protoroporphyrin IX. ChlH is acted upon by the ChlI:ChlD complex for magnesium insertion. This <i>Chlamydomonas</i> mutants with defects in this protein are chl1 and brs-1 and result in a brown phenotype. Transcription is also light regulated.
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<b><font size=3>Gun4</font size>
<b><font size=3>Gun4</font size>
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- Tetrapyrrole-binding protein</b>
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- Tetrapyrrole-binding Protein</b>
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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.  
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In <i>Arabidopsis</i>, GUN4 <i>(Genomes uncoupled 4)</i> 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 signalling by regulating magnesium-protoporphyrin IX synthesis or trafficking.
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<font size = 5><span style="color:#800000"><b>Mg-Protoporphyrin IX</b></font size> </span>
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<b><font size=3>ChlM</font size>
<b><font size=3>ChlM</font size>
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- Mg protoporphyrin IX S-adenosyl methionine O-methyl transferase</b>
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- Mg Protoporphyrin IX S-adenosyl Methionine O-methyl Transferase</b>
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Magnesium-protoporphyrin O-methyltransferase (chlM) [PMID: 12828371; PMID: 12489983; PMID: 4436384]; ChloroP 1.1 predicts cp location
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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 an enzyme called “ChlM - Mg protoporphyrin IX S-adenosyl methionine O-methyl transferase”.
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<font size = 5><span style = "color:#DC381F"><b>Mg-Proto ME</b></font size> </span>
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<b><font size=3>CTH1</font size>
<b><font size=3>CTH1</font size>
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- Copper target 1 protein</b>
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- Copper Target 1 Protein</b>
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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].  
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Functional variant produced under copper and/or oxygen sufficient conditions, Mg-protoporphyrin IX monomethyl ester (oxidative) cyclase.  
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<b><font size=3>Plastocyanin</font size>
<b><font size=3>Plastocyanin</font size>
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- Chloroplast precursor</b>
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- Chloroplast Precursor</b>
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pre-apoplastocyanin, PETE [PMID: 2165059; PMID: 8940133]; structure of plastocyanin PDB: 2PLT; mutant = ac208 [PMID: 8463310]
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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.
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<b><font size=3>YCF54</font size>
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- Oxidative Cyclase Cofactor</b>
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YCF54 is the second cofactor in oxidative cyclase.
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<font size = 5><span style="color:#52D017"><b>Protochlorophyllide</b></font size> </span>
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<b><font size=3>POR</font size>
<b><font size=3>POR</font size>
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- Light-dependent protochlorophyllidereductase</b>
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- Light-dependent Protochlorophyllide Reductase</b>
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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]
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A chloroplast precursor which converts protochlorophyllide to chlorophyllide using NADPH and light as the reductant.
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<font size = 5><span style="color:#41A317"><b>Chlorophyllide</b></font size> </span>
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<b><font size=3>DVR1</font size>
<b><font size=3>DVR1</font size>
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- 3,8-divinyl protochlorophyllidea 8-vinyl reductase</b>
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- 3,8-divinyl protochlorophyllide a 8-vinyl Reductase</b>
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Predicted chloroplast transit peptide 1-58; [PMID: 15695432; PMID: 15849308]
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Encodes for 3,8-divinyll Pchlide a 8-vinyl reductase that has important function in reduction of 8-vinyl groupto the ethyl group on tetrapyrrole using NADPH as substrate. In addition to that, it is also responsible in conversion of divinyl protochlorophyllide or a divinyl chlorophyllide to monovinyl protochlorophyllide a or monovinyl chlorophyllidevia reduction of vinyl group.
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<b><font size=3>ChlG</font size>
<b><font size=3>ChlG</font size>
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- Chlorophyllsynthetase</b>
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- Chlorophyll Synthetase</b>
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Catalyses the esterification of chlorophyllide with phytyl-pyrophosphate to make chlorophyll
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A nuclear encoded gene which encodes chloroplast transit sequences for translocation of enzymes into the chloroplast using specific substrates. <i>E.g.</i> Phytyl-pyrophosphate and geranylgeranyl-pyrophosphate are substrates used by <i>Avenasativa</i> chlorophyll synthase.
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<b><font size=3>ChlP</font size>
<b><font size=3>ChlP</font size>
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- Geranylgeranyl reductase</b>
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- Geranylgeranyl Reductase</b>
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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.
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Reduces the geranylgeranyl group to the phytyl group in the side chain of chlorophyll. Plant geranylgeranyl hydrogenase (ChlP) reduces free geranylgeranyl diphosphate to phytyl diphosphate, which provides the side chain to chlorophylls, tocopherols, and plastoquinones.
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<font size = 5><span style="color:#008000"><b>Chlorophyll a</b></font size> </span>
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<h1><center>Methods and workflow</center></h1>
<h1><center>Methods and workflow</center></h1>
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<center><h7> The methods we planned on using to recreate the pathway...(?)</h7></center>
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<center><h7><p> A quick summary of how we planned to approach the introduction of chlorophyll biosynthesis into <i>E. coli</i></p></h7></center>
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<b><font size=4>Design</font size></b>
<b><font size=4>Design</font size></b>
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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 <i>E. coli</i>.
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We designed 10 genes necessary for chlorophyllide biosynthesis, with 1 co-factor gene and another 2 genes for chlorophyll biosynthesis, totaling 13 genes. These genes were also codon optimised for expression within <i>E. coli</i>.
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<b><font size=4>Assembly</font size></b>
<b><font size=4>Assembly</font size></b>
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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.
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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.
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<b><font size=4>Transformation</font size></b>
<b><font size=4>Transformation</font size></b>
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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,
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,
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[[File:Construct11113.jpg|650px|thumb|center|Assembly of BioBricks via restriction enzyme digestion]]<html>
[[File:Construct11113.jpg|650px|thumb|center|Assembly of BioBricks via restriction enzyme digestion]]<html>
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<b><font size=4>Transformation & Characterizations</font size></b>
<b><font size=4>Transformation & Characterizations</font size></b>
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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.
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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 our BioBricks simultaneously.
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<h1><center>Highlighted results</center></h1>
<h1><center>Highlighted results</center></h1>
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<center><h7> The construction of the gene pathway... </h7></center>
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<center><h7><p> Shown here are some of our most important and successful results, summarized. Consult our <span style="color:#8B0000"><font size = 3><a class="three" href='https://2013.igem.org/Team:Macquarie_Australia/Results'><b>Results link</b></a></font size></span> for more information</center>
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<b>Gene Sequencing Results -</b> All of our genes have been shown to be ligated correctly from gBlocks, with all our sequencing results submitted, having comeback with an identity match of <b>100%</b>
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<br> <center><b>Twelve BioBricks</b> were successfully constructed<br>
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<img src=https://static.igem.org/mediawiki/2013/c/cc/Mq_biobrick_overview.jpg width = 500></center>
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<br> <center><b>Experimental verification of BioBrick function</b> (ChlD)<br>
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<center><img src = https://static.igem.org/mediawiki/2013/5/58/MQ_ChlD_2_4.jpg width =500></center>
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[Part 2-The Experiments-Part 3-Results]
 
<h1><center>Future/Significance of project</center></h1>
<h1><center>Future/Significance of project</center></h1>
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<center><h7> Back to the Future IVXC </h7></center>
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<center><h7> <p>Our research provides an innovative approach to plant synthetic biology with the potential
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to change the future of green energy and research on photosynthesis. As this is an initial step in eventually
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synthetically building the entire Photosystem II pathway, the potential to obtain
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hydrogen gas and channel the electrons into energy synthesis would be a major
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breakthrough in green energy. </p></h7></center>
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<b>Alternative Energy Source</b>
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The use of hydrogen gas as an energy source would provide many benefits to society,
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the environment and the economy. According to the United Nations Industrial
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Organisation, approximately three quarters of industrial energy use goes into the
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production of commodities that in turn cost more energy when consumed such as
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paper and metals. As these energy requirements are costly to businesses, an
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alternative energy source that was both cheap and efficient would be enticing to
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business owners. Furthermore, a reduction in carbon emissions as a result of this new
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technology would greatly improve the health of the environment. It is important that
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time and funds are invested into promising projects such as this one to ensure that the
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environment is protected from carbon emissions and pollution that are increasing as
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the demand for energy also rapidly increases.
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<b>Photosynthesis Research</b>
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Perhaps of most significance in the short term is the impact that the assembly all of
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the genes in the chlorophyll biosynthetic pathway will have on our understanding of
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how the system works. If the genes for chlorophyll can be effectively expressed in a
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non-photosynthetic bacterium then this will advance our current understanding of
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how to manipulate plant genes which has proven difficult in the past. Thus, this step is
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crucial in achieving the overall goal of harnessing a new source of green energy.
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<center><img src = https://static.igem.org/mediawiki/2013/b/bf/MQbulbsideways2.jpg></center>
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Latest revision as of 03:56, 28 September 2013




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

Background link
Results link


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 promoter operons based on function.

Production of a biosynthetic pathway and qualification assays of protein function






Figure - Our construction of three operons to synthesise chlorophyll from protophoryphyrin iX in E. coli.




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.



Smiley face

Protoporphyrin IX

Chll1 - Magnesium Chelatase subunit I
Catalyzes the insertion of magnesium ion into protoporphyrin IX to yield Mg-protoporphyrin IX. Forms an ATP dependent hexameric ring complex and a complex with the ChlD subunit. Transcript is light regulated and may be diurnal and/or circadian.

Chll2 - Magnesium Chelatase subunit I
The second gene which catalyzes the insertion of magnesium ion into protoporphyrin IX to yield Mg-protoporphyrin IX. Forms an ATP dependent hexameric ring complex and a complex with the ChlD subunit.

ChlD - Magnesium Chelatase subunit D
Forms an ATP dependent complex with the ChlI subunits 1 & 2, before acting on the protoporphyrin which is bound to the ChlH protein to insert magnesium.

ChlH - Magnesium Chelatase subunit H
Involved in bacteriochlorophyll pigment biosynthesis; introduces a magnesium ion into protoporphyrin IX to yield Mg-protoroporphyrin IX. ChlH is acted upon by the ChlI:ChlD complex for magnesium insertion. This Chlamydomonas mutants with defects in this protein are chl1 and brs-1 and result in a brown phenotype. Transcription is also light regulated.

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 signalling by regulating magnesium-protoporphyrin IX synthesis or trafficking.

Mg-Protoporphyrin IX

ChlM - Mg Protoporphyrin IX S-adenosyl Methionine O-methyl Transferase
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 an enzyme called “ChlM - Mg protoporphyrin IX S-adenosyl methionine O-methyl transferase”.

Mg-Proto ME

CTH1 - Copper Target 1 Protein
Functional variant produced under copper and/or oxygen sufficient conditions, Mg-protoporphyrin IX monomethyl ester (oxidative) cyclase.

Plastocyanin - Chloroplast Precursor
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.

YCF54 - Oxidative Cyclase Cofactor
YCF54 is the second cofactor in oxidative cyclase.

Protochlorophyllide

POR - Light-dependent Protochlorophyllide Reductase
A chloroplast precursor which converts protochlorophyllide to chlorophyllide using NADPH and light as the reductant.

Chlorophyllide

DVR1 - 3,8-divinyl protochlorophyllide a 8-vinyl Reductase
Encodes for 3,8-divinyll Pchlide a 8-vinyl reductase that has important function in reduction of 8-vinyl groupto the ethyl group on tetrapyrrole using NADPH as substrate. In addition to that, it is also responsible in conversion of divinyl protochlorophyllide or a divinyl chlorophyllide to monovinyl protochlorophyllide a or monovinyl chlorophyllidevia reduction of vinyl group.

ChlG - Chlorophyll Synthetase
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 Avenasativa chlorophyll synthase.

ChlP - Geranylgeranyl Reductase
Reduces the geranylgeranyl group to the phytyl group in the side chain of chlorophyll. Plant geranylgeranyl hydrogenase (ChlP) reduces free geranylgeranyl diphosphate to phytyl diphosphate, which provides the side chain to chlorophylls, tocopherols, and plastoquinones.

Chlorophyll a





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, with 1 co-factor gene and another 2 genes for chlorophyll biosynthesis, totaling 13 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,

Assembly of BioBricks via restriction enzyme digestion


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 our BioBricks simultaneously.





Highlighted results

Shown here are some of our most important and successful results, summarized. Consult our Results link for more information



Gene Sequencing Results - All of our genes have been shown to be ligated correctly from gBlocks, with all our sequencing results submitted, having comeback with an identity match of 100%


Twelve BioBricks were successfully constructed





Experimental verification of BioBrick function (ChlD)






Future/Significance of project

Our research provides an innovative approach to plant synthetic biology with the potential to change the future of green energy and research on photosynthesis. As this is an initial step in eventually synthetically building the entire Photosystem II pathway, the potential to obtain hydrogen gas and channel the electrons into energy synthesis would be a major breakthrough in green energy.


Alternative Energy Source
The use of hydrogen gas as an energy source would provide many benefits to society, the environment and the economy. According to the United Nations Industrial Organisation, approximately three quarters of industrial energy use goes into the production of commodities that in turn cost more energy when consumed such as paper and metals. As these energy requirements are costly to businesses, an alternative energy source that was both cheap and efficient would be enticing to business owners. Furthermore, a reduction in carbon emissions as a result of this new technology would greatly improve the health of the environment. It is important that time and funds are invested into promising projects such as this one to ensure that the environment is protected from carbon emissions and pollution that are increasing as the demand for energy also rapidly increases.

Photosynthesis Research
Perhaps of most significance in the short term is the impact that the assembly all of the genes in the chlorophyll biosynthetic pathway will have on our understanding of how the system works. If the genes for chlorophyll can be effectively expressed in a non-photosynthetic bacterium then this will advance our current understanding of how to manipulate plant genes which has proven difficult in the past. Thus, this step is crucial in achieving the overall goal of harnessing a new source of green energy.