Team:Macquarie Australia/Project/Parts

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Project


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

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.
Operon 2

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

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 5: 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
PartsTypeDescriptionDesignerLength
BBa_K1080002CodingChlDiGEM13_Macquarie_Australia2154
BBa_K1080003CodingGUN4iGEM13_Macquarie_Australia728
BBa_K1080006CodingPlastocyaniniGEM13_Macquarie_Australia324
BBa_K1080000CodingChlI1iGEM13_Macquarie_Australia1116
BBa_K1080001CodingChlHiGEM13_Macquarie_Australia4125
BBa_K1080005CodingCTH1iGEM13_Macquarie_Australia1152
BBa_K1080007CodingPORiGEM13_Macquarie_Australia1067
BBa_K1080008CodingChlPiGEM13_Macquarie_Australia1299
BBa_K1080009CodingChlGiGEM13_Macquarie_Australia1050
BBa_K1080010CodingYCF54iGEM13_Macquarie_Australia471
BBa_K1080011CodingChlI2iGEM13_Macquarie_Australia1212
BBa_K1080012CodingDVR1iGEM13_Macquarie_Australia1106
BBa_K1080013CompositeGUN4 (+ Ptac Promoter)iGEM13_Macquarie_Australia797
BBa_K1080014CompositePlasto (+ Ptac promoter)iGEM13_Macquarie_Australia393
BBa_K1080015CompositePOR (+ Ptac promoter)iGEM13_Macquarie_Australia1136
BBa_K1080016CompositeChlI1 (+Ptac promoter)iGEM13_Macquarie_Australia1185
BBa_K1080017CompositeChlH (+ Ptac promoter)iGEM13_Macquarie_Australia4194
BBa_K1080018CompositeChlD (+ Ptac promoter)iGEM13_Macquarie_Australia2223
BBa_K1080019CompositeChlM (+ Ptac promoter)iGEM13_Macquarie_Australia942
BBa_K1080020CompositeCTH1 (+ Ptac promoter)iGEM13_Macquarie_Australia1221
BBa_K1080021CompositeChlP (+ Ptac promoter)iGEM13_Macquarie_Australia1368
BBa_K1080022CompositeChlG (+ Ptac promoter)iGEM13_Macquarie_Australia1119
BBa_K1080023CompositeYCF54 (+ Ptac promoter)iGEM13_Macquarie_Australia540
BBa_K1080024CompositeChlI2 (+ ptac promoter)iGEM13_Macquarie_Australia1281
BBa_K1080025CompositeDVR1 (+ Ptac promoter)iGEM13_Macquarie_Australia1175
BBa_K864400RegulatoryPtac, trp & lac regulated promoteriGEM12_Uppsala61

Parts improved

Part NameTypeDescriptorLength
BBa_K1080002Singular/CodingChlD2154

Parts Submitted by Team Macquarie Australia 2014

<groupparts>iGEM014 Macquarie_Australia</groupparts>

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