Team:Macquarie Australia/Demonstrate

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Results

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Fig 1. From left to right - hemH mutant cells, hemH mutant cells with Mg-chelatase operon, control DH5a cells in order of 0mM, 2mM and 5mM for each set of three. From top to bottom: day 1, day 3, day 5 and day 7. We can visually see the colour change from cloudy and opaque to more red due to the conversion of ALA to PPIX.

Our tubes of the original 45 samples did not have consistent colouring as we would have expected. Day 5 samples appeared to be more red in colour than day 7 samples.

Fig 2. From top to bottom - day 1, day 3 and day 5 cells of the Mg-chelatase plasmid + mutant cells + IPTG. The day 5 cells are noticeably darker than both day 1 and day 3, and the day 5 0mM ALA sample.

Fig 3. a) CPIII absorbance readings for the DH5a control cells. b) PPIX absorbance readings for the DH5a control cells. c) CPIII absorbance readings for the hemH mutant cells. d) PPIX absorbance readings for the hemH mutant cells. e) CPIII absorbance readings for the hemH mutant with Mg-chelatase plasmid cells. f) PPIX absorbance readings for the hemH mutant with Mg-chelatase plasmid cells. g) CPIII absorbance readings for the hemH mutant with Mg-chelatase plasmid induced with IPTG. h) PPIX absorbance readings for the hemH mutant with Mg-chelatase plasmid induced with IPTG. Control cells are visually lower in both Protoporphyrin IX and Coproporphyrin III production than the three mutant colonies.

Fig 4. Graph of the 590nm fluorescence output for the Mg-chelatase plasmid with mutant and IPTG. MgPPIX concentration increases after day 3.

Discussion

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Our graphs of each colony are very distinguishable across the varying ALA concentrations and liquid cultures. We can recognise that the control culture of DH5a cells are much less efficient at producing CPIII and PPIX than the colonies containing the hemH mutant. On the same scale, the control has negligible amounts of both CPIII and PPIX. The experimental results revealed an additional bottleneck for maximum flux through the pathway at the penultimate step. CPIII is the intermediate in the chlorophyll biosynthesis pathway before the generation of PPIX, therefore, it was produced faster over the first few days before PPIX started to appear. The rates of formation were consistent with the model. As expected, our 0mM ALA tubes did not produce a significant amount of PPIX, and the highest concentration of ALA at 5mM produced the highest yield of both CPIII and PPIX.

We observe the increase in MgPPIX concentration at day 3 in figure 4 above. According to figure 3, that is when PPIX is starting to be generated. We therefore conclude that we only get accumulation of MgPPIX when PPIX increases within the cell and when we induce with IPTG.

The inconsistent colouring of the tubes was puzzling to us. We centrifuged the original samples to observe the pellet size, as we thought differing amounts of substances may have an effect on the colour. We observed different pellet sizes, but did not analyse the composition of each. Our fluorescence results indicated that the hemH mutant was effective at producing PPIX compared to the wildtype. The experiments done in previous years extracted the whole culture, whereas this year we only extracted the cells and this indicated that PPIX was present in the cells of the mutant but not in the cells of the wildtype. We may suggest in the future that the pellets are analysed to determine whether the compositions are affecting the resulting output.

Fig 5. Pellets of each of the day 1 cells. From left to right - hemH mutant cells, hemH mutant cells with Mg-chelatase operon, control DH5a cells.

Fig 6. Pellets of each of the day 3 cells. From left to right - hemH mutant cells, hemH mutant cells with Mg-chelatase operon, control DH5a cells.

Fig 7. Pellets of each of the day 5 cells. From left to right - hemH mutant cells, hemH mutant cells with Mg-chelatase operon, control DH5a cells.

Fig 8. Pellets of each of the day 7 cells. From left to right - hemH mutant cells, hemH mutant cells with Mg-chelatase operon, control DH5a cells.

Fig 9. Day 1 Mg-chelatase plasmid + mutant + IPTG pellet sizes.

Fig 10. Day 3 Mg-chelatase plasmid + mutant + IPTG pellet sizes.

Fig 11. Day 5 Mg-chelatase plasmid + mutant + IPTG pellet sizes.

Conclusions

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We can confirm that the rates of formation of the intermediates, namely PPIX, were consistent with our modelling. The highest concentration of ALA (5mM) produced the most CPIII and PPIX, and the lowest concentration (0mM) produced negligible amounts of both intermediates. These results also support Macquarie's 2015 iGEM modelling results for the production of PPIX.

Future Experimental Considerations

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For future experiments, we suggest:
• Analysing the pellet composition to determine whether the compositions are affecting the resulting output
• Altering the media and growth conditions utilised by the experiment to provide alternative results that should still support our hypothesis
• Running the IPTG induced cells for 7 days for an accurate comparison between the liquid cultures
• Repeating the whole experiment a couple more times for accuracy
• Test the pathway all the way from ALA to chlorophyll a, as that is the new main model

Looking To The Future

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In terms of creating exciting simulations and simple graphs that could be a model in the future, here is a list of ideas that might have the opportunity to be performed:

Improve current estimation and application of models to take a step further in reaching our goal:
• Increase efficiency of hydrogen production via trial simulations with different environmental factors.
• For example, Mg-chelatase plasmid is designated as a whole system throughout the chlorophyll biosynthesis and hydrogen production pathway.
• Production yield can be maximised by testing and adjustment of limiting parameters or further investigation on other alternatives.

Create an application or program that provides a life interactive model of the chlorophyll biosynthesis pathway:

• It will visually communicate the knowledge of functional intermediates, substrates and enzymes within the pathway especially towards education and outreaching the public.
• How different parameters were set and the effect it has on the biosynthesis pathway.
• Assist pilot studies such as a comparison between different environments in which the biosynthesis pathway functions (being in a E. coli or Chlamydomonas cell ).

The concept design becomes an actual, working prototype:

• Engineering and computational skills will be put to the test to create a living cell prototype of this year’s concept design.
• Many estimations or numbers will be made on functions, circuits and dimensional design of the new prototype.
• Risk assessments also could be made for the prototype to be available for public use, especially for farmers.
• Overall, it bring 2D ideas to 3D actions, providing future opportunities that may impact the markets, biotech industries and everyday life.
  
  Uncertainties:
  
• As new ideas and concepts come into view, a whole new direction can lead to unforeseen circumstances.




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