Following is an overview of the results given from our work with industry and community

1-2 paragraph overview of work done should link to pages and specific areas if mentioned

1-2 paragraph overview of work done should link to pages and specific areas if mentioned

1-2 paragraph overview of work done should link to pages and specific areas if mentioned

1-2 paragraph overview of work done should link to pages and specific areas if mentioned

Following is an overview of the results gained in the Wet Lab for the 2019 iGEM Competition

• Golden gate assembly was used to assemble six constructs to express the 6GIX protein, containing each of the different signal peptides OmpA, MalE, TorA, YcbK, DsbA, and PhoA and cloned into pSB1A3.
• The 6GIX protein was expressed under IPTG induction conditions and purified using Ni-NTA chromatography. The correct protein size was visible at 21 kDa. Further confirmation of protein expression was evidenced from western blotting.
• The 6GIX protein was secreted using the signal peptides PhoA, DsbA, and MalE, with the brightest protein band visible for secretion using the MalE signal peptide.

1-2 paragraph overview of work done should link to pages and specific areas if mentioned

1-2 paragraph overview of work done should link to pages and specific areas if mentioned

Ultimately, pheophorbide a was shown to have an inhibitory effect on the mycelial growth rate of Sclerotinia sclerotiorum (Figure 2B, 5A, 6B, 9) and had no visible effect on Pestalotiopsis microspora (Figure 3, 5B, 7). This inhibitory effect was proven to be controlled by photo-activation and was positively correlated with increasing treatment concentrations (Figure 2B, 5A, 6B, 9). Recorded inhibition of Sclerotinia sclerotiorum under varying conditions, suggests that the time of pheophorbide a photo-activation, relative to the treatment discs' distance to the culture is important. Particularly, the compound may need to be reapplied if there was insufficient pheophorbide a application initially, which may be a result of diffusion. It was also shown that with increasing light exposure there is a significant decrease in Sclerotinia sclerotiorum mycelial growth (Figure 4A) and a marginal increase in Pestalotiopsis microspora mycelial growth rate (Figure 4B). These results provide support for the use of pheophorbide a as an anti-fungal agent and provide preliminary grounding for its use as a preventative measure against Sclerotinia sclerotiorum infection.

Figure 6A and 6B. Mycelial growth of Sclerotinia sclerotiorum with pheophorbide a in dark (6A-left) and in light (6B-right) conditions. Five treatments were applied as treatment discs impregnated with pheophorbide a, solubilized in 25% acetone (0, 5, 15, 25, 35 mg/mL). The 0 mg/mL treatment was 25% acetone. Two treatment discs for each concentration were placed 1.5 cm from the epicentre of the fungal culture. Growth was tracked for four days after culturing. Measurements were taken once a day from the epicentre of the original culture to the edge of the mycelial growth toward the disc. Growth is maxed at 4.2 cm due to the potato dextrose agar plate capacity. Light conditions were done using 1400 lumen white LED light at a distance of 25 cm from the plate. Data points are summarized in Anti-Fungal Table 6A and 6B here.

The main objective for our exploration of pheophorbide a as an anti-fungal agent is for it to become a usable preventative measure to counter Sclerotinia Sclerotiorum infection. To do this we plan on further replication of mycelial growth inhibition experiments, while also furthering these by testing the effect of reapplying pheophorbide a each day. We would like to determine the minimum inhibitory concentration of our compound relative to fungal pathogens by further dosage testing and perform chemical-genetic profiling to determine the mode of action of pheophorbide a as a drug compound. Finally, we would like to perform whole organism tests with canola plants to test pheophorbide a's ability to prevent a fungal infection after Sclerotinia sclerotiorum spores have landed on the plant's leaves. You can read more about our future directions for pheophorbide a here.

Following is an overview of the results gained in the Dry Lab for the 2019 iGEM Competition

1-2 paragraph overview of work done should link to pages and specific areas if mentioned

1-2 paragraph overview of work done should link to pages and specific areas if mentioned

1-2 paragraph overview of work done should link to pages and specific areas if mentioned

1-2 paragraph overview of work done should link to pages and specific areas if mentioned

The BioBrick Optimization Tool for synthesis was shown to be successful, as it could generate sequences with a lower idt score for the SGR sequence.