Team:Macquarie Australia/Results
Protochlorophyllide was extracted from barley was used as a substrate for lysate of E.coli containing trc-POR induced by IPTG. The mixture was left for X time then placed in a spectrophotometer. The absorbance given by protochlorophyllide was able to be clearly seen but unfortunately absorbance peaks for chlorophyllide (product given by POR) were unable to be seen. We believe this may be due to the fact that POR is a membrane bound protein and that would not have been able to function with protochlorophyllide after lysis of the cells.
All composite parts underwent single (EcoRI) and double (EcoRI with PstI) digests followed by agarose gel (0.5%) electrophoresis to summarise and validate the successful construction of the parts comprising the Chlorophyll Biosynthesis Plasmid (See figure 1).
- 1. Assembly- Mg-Chelatase plasmid (trc-ChlI1-ChlI2-ChlD-trc-ChlH-GUN4), Protoporphyrin IX (PPIX) Chelator
This biobrick was created to begin the 1st step of the Chlorophyll biosynthesis pathway away from Heme biosynthesis utilising the cells naturally produced PPIX to Mg-PPIX. Standard assembly of trc-ChlI1-ChlI2-ChlD and trc-ChlH-GUN4 was performed in a CAM backbone. - 2. Assembly- trc-POR-ChlP-DVR1-ChlG (PPDG), Protochlorophyllide (PChlide) to Chlorophyll A biosynthesis
The PPDG biobrick was created as the final part of our Chlorophyll A biosynthesis created to convert photoxidative PChlide to Chlorophyll A to allow stability of our light triggered vesicle formation system. This part was formed using Standard assembly of trc-POR-ChlP and DVR1-ChlG into a CAM backbone. - 3. Assembly- Chlorophyll A Biosynthesis Plasmid (CBP)
The CBP Biobrick pieced together all 14 genes of our Chlorophyll biosynthesis pathway into a single 18kb Biobrick in a CAM backbone. This was formed using Standard assembly of previously assembled Mg-Chelatase plasmid and lac-CTH1-ycf54-ChlM-trc-FNR-fdx-trc-POR-ChlP-DVR1-ChlG.
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