Results

Here, we have re-stated our results which were initially discussed in our Engineering Success and Proof of Concept pages.

Engineering Success - Results

See the Engineering Success Page to read these results in context

The last part of confirming Engineering Success was to test and validate the gene expression of our construct. The assay that validated the effectiveness of our biopesticide revolved around identifying Cyt1Aa and Cyt2Ba’s efficacy in reducing the population of Dipteran insect larvae. We used wingless Drosophila melanogaster as the model insect population in our project, however other insect pests that fall under the order Diptera, such as Fungus gnats, leafminers, and shore files can also be applied to this project model. We modelled the functions of Cyt1Aa and Cyt2Ba through a cytotoxicity assay. Additionally, we wanted to characterise fluorescence of the strains as further on we hope to attempt a root colonization assay in our Proof of Concept and will image successful colonization using fluorescent microscopy.

Cytotoxicity Assay

The Cyt1Aa- and Cyt2Ba-containing plasmids (pCG004) will be induced in a B. subtilis culture. The culture was then used to soak cotton pads that were introduced to the D. melanogaster population (Figure 4) and the insects were scored dead if they are no longer moving for a few minutes and alive if they are moving continuously which was then recorded to quantify the percentage of mortality. Based on our preliminary literature review, we expected to see a decrease in the population size in the treatment conditions. Although we have changed the target species that the initial paper used, we still expect to observe a population decrease because D. melanogaster is within the Dipteran order.

Figure 4. Set up of the Cytotoxicity Assay. Cyt1Aa- and Cyt2Ba-expressing B. subtilis and E. coli strains were soaked into cotton and had D. melanogaster exposed to it for 10 minutes and death was recorded.

For the parameters of the assay flies were separated into three trials using: B. subtilis empty backbone, B. subtilis-Cyt1Aa, B. subtilis-Cyt2Ba, E. coli-Cyt1Aa and E. coli-Cyt2Ba. We included our E. coli mutants to test if gene expression of the Cyt proteins could be expressed at notable levels even in lab strains. This is touching on our values of accessibility as we want as many iGEM teams to be able to address proof of concept biopesticide use in common lab strains (as E. coli and D. melanogaster are common model organisms, more so that B. subtilis and fungal gnats). After 10 minutes of exposure to the mutant strains, their killing activity was quantified using excel by the number of dead flies (Table 2) and the data plotted in the group below using Prism Graphpad Software (Figure 5).

Table 2.Results from the Cytotoixicty Assay
Raw Numbers Replicate B.subtilis Empty Backbone B.subtilis Cyt1Aa B.subtilis Cyt2Ba E.coli Cyt1Aa E.coli Cyt2Ba
# of D. melanogaster killed 1 0/3 2/3 7/7 1/8 2/5
# of D. melanogaster killed 2 0/4 1/3 4/5 2/4 3/6
# of D. melanogaster killed 3 1/3 3/3 1/3 2/3 2/4
Total Killed - 1 6 12 5 7
Total Tested - 10 9 15 15 15
Percentage Killed (%) 1 0 66 100 12.5 40
Percentage Killed (%) 2 0 33 80 50 50
Percentage Killed (%) 3 33 100 33 66 50
Average Percentage Killed (%) - 11 66.3 71 42.8 46.7

Figure 5. Toxicity assay trials of Cyt-containing plasmids. Measurements were taken at the 10 minute mark and the average percentage of killed D. melanogaster was plotted finding the mean from triplicate trials and the Standard Error of the Mean (SEM) was calculated and shown in the error bars. Strain names can be found in Table 1 and the raw data for this graph can be found in Table 2.

Fluorescence Testing

For imaging and quantification of B. subtilis colonization of tomato plant roots, the fluorescent markers needed to be tested in the B. subtilis-Cyt strains (Table 1) and the B. subtilis controls. The strains were imaged in two experiments under conditions that induce fluorescence to test it.

UV Induced Fluorescence

The first test was simply imaging 5 mL cultures grown for 18 hours using a UV-wand to see levels of fluorescence that occurred (Figure 6).

Figure 6. Fluorescence of B. subtilis-Cyt strains when observed under UV light. The strains in order of Left to Right are as follows (A) B. subtilis WT, (B) B. subtilis Empty Bacbone (C)B. subtilis Cyt1Aa, (D)B. subtilis Cyt2Ba.

While the fluorescent colours of GFP, mScarlet and BFP could not be specifically determined, fluorescence was seen in all of our strains that could fluoresce while our WT did not fluoresce. This meant that we could continue to our next experiment, visualising fluorescence using a microscope.

Microscopic Observation of Fluorescence

The second test was one conducted using a microscope to verify the fluorescence of the four B. subtilis strains. Briefly, overnight strains were diluted to OD 0.2 in SD media with ITPG to induce gene expression. They were grown for 1 hour and imaged using the relevant filters in a 96-well plate using a Nikon microscope (Figure 7).

Figure 7.B. subtilis Cyt1Aa fluorescing red due to mScarlet expression as an example image. While B. subtilis Cyt1Aa was used as an example image, all of the other strains had images that demonstrate that the strains do indeed fluoresce the correct colours. From the images it can be seen that the strains do indeed fluoresce the correct colours.

Proof of Concept - Results

See the Proof of Concept Page to read these results in context

Proof of Concept results are given throughout, since we did experiments for this in a few major categories. Results are at the end of each major category, so viewing the proof of concept results in context is recommended. Thank you for your interest, we hope you'll go check it out!

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