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.

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.

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''.

The ChlD Story: the repair of the registry chlD

Team:Macquarie_Australia_2013 designed and synthesized a number of parts that would be used for the biosynthesis pathway. Among these, they designed and synthesized chlD, Bba_K1080002. This part was not recieved by the registry in 2013.

As we required this part for the assembly of our composite operons, we verified the identity and sequence of all of the parts. When sequencing chlD, we determined that there was a 50-basepair deletion in the middle of chlD. Team_Macquarie_2014 has repaired this part. We incorporated it into operon 1, and have verified that the part is functional and therefore 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:Macquarie_Australia_2013. Again, many of these were not received by the registry. Following our efforts to recheck the DNA sequence, we improved their documentation to further elucidate the interactions between each part of the system. We therefore added information about their function, structure and enzymatic reactions, which may be found in the parts registry.