Team:Macquarie Australia/Project/Overview

We have engineered the essential genes necessary for the biosynthesis of chlorophyll a in E. coli and showed functionality as well as modelled the first step in the pathway.

The project aim is to engineer the essential genes necessary for the synthesis of chlorophyll a in E. coli. This is first part of the overall goal with the construction of photosystem II and a artificial photosynthesis to generate hydrogen gas for energy production. The construction of 3 operons containing 11 of the 12 essential genes in the pathway has been performed in our project (Fig. 1). A previously assembled biobrick (ChlD; BBa_K1080002) containing a 50 bp deletion was also repaired and re-characterised on the registry. We have demonstrated functionality (as see in Results) of the part of the first operon (Mg-chelatase) and have also modelled this stage within the chlorophyll biosynthesis pathway (as seen in Modelling).

Figure 1. Operon composites parts constructed in their respective steps in the chlorophyll a synthesis pathway. Assembly was performed using the restriction digest and ligation protocol. All operons contain the lac promoter for subsequent induction for functionality assays.

Assembly of the 3 operons was performed through a restriction digest and ligation protocol (link to ligation protocol), confirmed through gel electrophoresis, DNA sequencing and mass spectrometry of the expressed proteins (as see in Results). The functionality of the first operon, Mg-chelatase, was demonstrated through the spectral analysis of its enzymatic product, Mg-protophoryin IX (link to results). This step of the enzymatic pathway was also modelled (as seen in Modelling).

Figure 2. Fluorescence spectra at 420 nm excitation of induced Chli1+ChlD (BBa_K1326004) extract following incubation in Figure 1. Peak observed at 595nm is consistent with Mg-Protoporphyrin IX production, demonstrating significant activity of the submitted BioBrick part.

The project was successful in building the foundations for future teams to complete the synthesis of photosystem II in E. coli. This provides a significant leap into the development of a hydrogen generating bacterial system and a renewable biological energy source.