Team:Macquarie Australia/Experimental
Introduction
Abbreviations
- ALA - aminolevulinic acid
- CPIII - coproporphyrin III
- PPIX - protoporphyrin IX
After generating our theoretical ALA-protoporphyrin model and ALA-Mg-protoporphyrin model, we needed to test them experimentally. Protoporphyrin IX (PPIX), one important precursors in the chlorophyll biosynthesis pathway, is also utilised in the production of heme in E. Coli. Ferrochelatase (encoded by hemH) is the last enzyme in the heme pathway, and responsible for the conversion of PPIX to heme. Therefore, by creating a dysfunctional ferrochelatase, we expect to interrupt the heme pathway, and thus push more PPIX into our chlorophyll pathway. For our experiment, we developed a mutant hemH E. coli strain that would theoretically be incapable of producing heme, and generating more PPIX instead. We visually observed the conversion of ALA to PPIX through a colour change from creamy to red. We also quantitatively assessed our experiment through spectrofluorescence.
Aim
To measure the production of protoporphyrin IX and Mg-protoporphyrin IX based on the parameters of our models.
Method
We produced four liquid cultures, one comprised of the mutant hemH E. coli strain, one with hemH and our Mg-cheletase plasmid, on with the hemH mutant, Mg-chelatase plasmid and IPTG, and the last as a control with competent DH5a E. coli cells. For each E. coli , we had ALA concentrations of 0mM, 2mM and 5mM as well as four separate tubes for removal on days 1, 3, 5 and 7 resulting in 45 tubes total (Mg-chelatase plasmid + IPTG had only 5 days). When the cultures had an optical density of approximately 0.8, we added components following the table below:
We added IPTG for induction of the Mg-chelatase enzyme components from the Mg-cheletase plasmid. This would ensure that the lac operon was induced and transcribed the genes necessary for the chlorophyll biosynthesis pathway. We added AMP as the cells were grown with ampicillin resistance, ensuring that only the cells we wanted grew and any others that were present were eradicated.
After incubation for the week, we analysed the colour changes of our colonies visually from a creamy, opaque colour to a red colour due to the presence of PPIX. We resuspended each sample and took 50μl and added 450μl of methanol and centrifuged on high speed. We measured spectrofluorescence of each of the supernatants at wavelengths of 404nm for PPIX and 420nm for Mg-PPIX. Data was obtained for both wavelengths and graphs were produced plotting wavelengths 620nm (for CPIII) and 630nm (for PPIX) on GraphPad Prism. For the Mg-chelatase plasmid induced with IPTG, we produced a graph showing the emission values at an excitatory wavelength of 590nm to generate a graph showing MgPPIX.
