Team:Macquarie Australia/Project/Parts

Parts & Characterization

The Macquarie 2014 team designed and constructed the following three operons required for the chlorophyll biosynthesis pathway. These three operons have been sent to the registry.

Functional Operons

Operon 1: BBa_K1326008
In anaerobic bacteria, the bch1 and bchD genes are part of an operon. Macquarie 2014 have constructed an equivalent synthetic operon in E. coli, using separate ChlI1 and ChlD taken from oxygenic photosynthetic eukaryotes. We have shown that our artificial operon works, and that proteins self-assemble to form a functional ChlI1:ChlD complex in E. coli. This part works together with ChlH to insert magnesium into protoporphyrin IX. Although our operon does not contain ChlH as originally planned (due to issues in fully assembling the part), in vitro assays indicated full functionality of self-assembly and catalytic functionality.

Figure 1. Operon 1, made up of the lac promoter, ChlI1, ChlD, and GUN4. The BioBrick plasmid backbone encodes chloramphenicol resistance.

Figure 2. Schematic representation of the conversion of Protophoryrin IX to Mg-Protophoryrin IX via Mg-chelatase (Operon 1). The functionality of this step has been demonstrated in our project.

Operon 2: BBa_K1326002
This operon has been constructed with genes responsible for catalysing the biosynthesis pathway from Mg-protoporphyrin IX to Protochlorophyllide. CTH1, Plastocyanin, and YCF54 are involved in the oxidative cyclase pathway. ChlM methylates Mg-protoporphyrin IX, facilitating the highly-regulated catalysis of Mg-chelatase. CTH1 catalyses the conversion of Mg-protoporphyrin IX monomethyl into divinyl protochlorophyllide, interacting with YCF54 and Plastocyanin.

Figure 3. Operon 2, made up of the lac promoter, CTH1, YCF54, Plasto, and ChlM. The BioBrick plasmid backbone encodes chloramphenicol resistance.

Figure 4. Schematic representation of the conversion of Mg-Protophoryrin IX to Divinyl protochlorophyllide via the genes in Operon 2.

Operon 3: BBa_K1326003
The construction of this operon included the genes responsible for the terminal steps of the chlorophyll biosynthesis pathway, in the conversion of divinyl protochlorophyllide to chlorophyll a. DVR1 reduces divinyl protochlorophyllide, POR converts protochlorophyllide to chlorophyllide, ChlG adds the geranylgeranyl pyrophosphate chain to the chlorophyllide molecule, and ChlP reduces the double bonds on GGPP. The final product is chlorophyll a.

Figure 5. Operon 3, made up of the lac promoter, POR, DVR1, ChlP, and ChlG. The BioBrick plasmid backbone encodes chloramphenicol resistance.

Figure 6. Schematic representation of the conversion of Divinyl protochlorophyllide to the final product of chlorophyll a. Operon 3 encodes the genes required for this conversion.

Parts Annotated and Improved

Last years' 2013 Macquarie iGEM team designed all 13 parts required for the biosynthesis pathway and successfully synthesized most of these. This year we screened and verified the identity of all their parts, as we required them to assemble composite parts with the aim of forming functional operons.

The ‘parts from previous teams used’ table below show the individual parts designed and synthesised by the 2013 Macquarie team, as well as the composite parts they made using each part with the lac promoter synthesised by the 2012 iGEM Uppsala team.

Many of the individual parts from last year were not received by the registry in 2013. Following our efforts to confirm all nucleotide sequences of parts, we thoroughly researched these genes and improved their documentation to elucidate the function and interactions between each part in the system. Information regarding each part's function, role in the biosynthesis pathway, protein structure, and enzymatic reactions can be found in the iGEM parts registry.

The ChlD Story: the repair of the registry ChlD

During our screening of all of last year’s individual parts, we discovered that the ChlD part [BBa_K1080002] had a 50 base-pair deletion in the middle of the gene.

We resolved this issue, repairing the deletion. With the complete and correct sequence present, this part could be incorporated into operon 1. We have since verified that the part is functional, guaranteeing it has been properly repaired.

This part has now been successfully sent to the registry in a functional state, and can be seen below in the parts improved table.

Parts from previous teams used

Parts improved

Parts Submitted by Team Macquarie Australia 2014

Favourite	Part Name	Type	Description	Designer	Length
	BBa_K1326000	Composite	Plac + CTH1 + YCF54	iGEM14_Macquarie_Australia	1700
	BBa_K1326004	Composite	Plac + ChlI1 + ChlD	iGEM14_Macquarie_Australia	3347
	BBa_K1326008	Composite	Plac + ChlI1 + ChlD + GUN4	iGEM14_Macquarie_Australia	4083
	BBa_K1326001	Coding	ChlM	iGEM14_Macquarie_Australia	867
	BBa_K1326002	Composite	Plac + CTH1 + YCF54 + Plasto + ChlM	iGEM14_Macquarie_Australia	2913
	BBa_K1326003	Composite	Plac + POR + DVR1 + ChlP + ChlG	iGEM14_Macquarie_Australia	4615
	BBa_K1326005	Composite	Plac + CTH1 + YCF54 + Plasto	iGEM14_Macquarie_Australia	2032
	BBa_K1326006	Composite	Plac + POR + DVR1	iGEM14_Macquarie_Australia	2250
	BBa_K1326007	Composite	Plac + POR + DVR1 + ChlP	iGEM14_Macquarie_Australia	3557