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 gene has been used in an operon with other 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.

Operon 3:BBa_K1326003
This gene has been used in an operon with other 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 4. Operon 3, made up of the lac promoter, POR, DVR1, ChlP, and ChlG. The BioBrick plasmid backbone encodes chloramphenicol resistance.

The ChlD Story: the repair of the registry ChlD

Team:iGEM2013_Macquarie_Australia designed all 13 parts required for the biosynthesis pathway and successfully synthesized most of these, including ChlD [BBa_K1080002.]. However, this part was not received by the registry in 2013.

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. When ChlD was sequenced, we determined that there was a 50 base-pair deletion in the middle of the gene. Team:iGEM2014_Macquarie_Australia has repaired this part, ensuring the full correct sequence is present. We incorporated this part it into operon 1, and have 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.

Parts Annotated and Improved

The following parts were again designed and synthesized by Team:iGEM2013_Macquarie_Australia. Many of these were not received by the registry in 2013. Following our efforts to confirm the DNA sequences, we improved their documentation to further elucidate the interactions between each part of the system. Information regarding each part's function, role in the biosynthesis pathway, protein structure, and enzymatic reactions, all of which can be found in the iGEM parts registry.

Parts from previous teams used

Parts	Type	Description	Designer	Length
BBa_K1080002	Coding	ChlD	iGEM13_Macquarie_Australia	2154
BBa_K1080003	Coding	GUN4	iGEM13_Macquarie_Australia	728
BBa_K1080006	Coding	Plastocyanin	iGEM13_Macquarie_Australia	324
BBa_K1080000	Coding	ChlI1	iGEM13_Macquarie_Australia	1116
BBa_K1080001	Coding	ChlH	iGEM13_Macquarie_Australia	4125
BBa_K1080005	Coding	CTH1	iGEM13_Macquarie_Australia	1152
BBa_K1080007	Coding	POR	iGEM13_Macquarie_Australia	1067
BBa_K1080008	Coding	ChlP	iGEM13_Macquarie_Australia	1299
BBa_K1080009	Coding	ChlG	iGEM13_Macquarie_Australia	1050
BBa_K1080010	Coding	YCF54	iGEM13_Macquarie_Australia	471
BBa_K1080011	Coding	ChlI2	iGEM13_Macquarie_Australia	1212
BBa_K1080012	Coding	DVR1	iGEM13_Macquarie_Australia	1106
BBa_K1080013	Composite	GUN4 (+ Ptac Promoter)	iGEM13_Macquarie_Australia	797
BBa_K1080014	Composite	Plasto (+ Ptac promoter)	iGEM13_Macquarie_Australia	393
BBa_K1080015	Composite	POR (+ Ptac promoter)	iGEM13_Macquarie_Australia	1136
BBa_K1080016	Composite	ChlI1 (+Ptac promoter)	iGEM13_Macquarie_Australia	1185
BBa_K1080017	Composite	ChlH (+ Ptac promoter)	iGEM13_Macquarie_Australia	4194
BBa_K1080018	Composite	ChlD (+ Ptac promoter)	iGEM13_Macquarie_Australia	2223
BBa_K1080019	Composite	ChlM (+ Ptac promoter)	iGEM13_Macquarie_Australia	942
BBa_K1080020	Composite	CTH1 (+ Ptac promoter)	iGEM13_Macquarie_Australia	1221
BBa_K1080021	Composite	ChlP (+ Ptac promoter)	iGEM13_Macquarie_Australia	1368
BBa_K1080022	Composite	ChlG (+ Ptac promoter)	iGEM13_Macquarie_Australia	1119
BBa_K1080023	Composite	YCF54 (+ Ptac promoter)	iGEM13_Macquarie_Australia	540
BBa_K1080024	Composite	ChlI2 (+ ptac promoter)	iGEM13_Macquarie_Australia	1281
BBa_K1080025	Composite	DVR1 (+ Ptac promoter)	iGEM13_Macquarie_Australia	1175
BBa_K864400	Regulatory	Ptac, trp & lac regulated promoter	iGEM12_Uppsala	61

Parts improved

Part Name	Type	Description	Designer	Length
BBa_K1080002	Singular/Coding	ChlD	iGEM13_Macquarie_Australia	2154

Parts Submitted by Team Macquarie Australia 2014

<groupparts>iGEM014 Macquarie_Australia</groupparts>