Summary
Troygenics - the platform system we developed during our project - possess the potential to be useful in
a vast range of applications. Due to the short timeframe of an iGEM project we only had the possibility to develop and test our
new system in the model organism S. cerevisiae. But the proof of our concept opens the door for so many
more conceivable applications.
As one possibility we focused on fighting plant pathogenic fungi with the aid of our Troygenics. To compare our system with
the commonly available treatments we investigated the impact of two different fungicides on our model organisms S. cerevisiae
and A. niger.
Additionally, we did some research on possible applications in nature and medicine and talked to several experts, that
called our Troygenics a promising approach.
As we were able to demonstrate that every subproject worked out as we expected, we finally assembled a complete
Troygenic. The generated experience from the subprojects Troygenic construction, endocytosis and CeDIS were merged
to a S. cerevisiae-specific Troygenic that presents mating factor alpha on its coat and codes for sfGFP, as well as
TRP1, a marker gene for tryptophane-auxotrophic yeast strains. We successfully expressed and purified the Troygenics
and were even able to start some initial experiments. WAS SAGT DIE COL-PCR?
But unfortunately, due to the time limits, we were not able to examine the optimal transformation conditions for
our Troygenics. This is a very interesting aspect, that could be investigated in the future. The most important fact
that should be determined is the optimal Troygenic-concentration for efficient transformation of the target cells.
Additionally, further experiments regarding the specificity of our Troygenics by comparing the
transformation-efficiency in the target cell and a closely related non-target cell would be of great interest.
Due to the fact that our project mainly focused on fighting plant-pathogenic fungi, it would additionally be
important to compare our system to conventionally utilized fungicides. Therefore, we investigated the effect of
different fungicides on our model organisms S. cerevisiae and A. niger.
Fungicide Simulation
As a part of our project, we decided to test the effects of two fungicides Folicur® and Proline® from Bayer AG to our model organisms Aspergillus niger and Saccharomyces cerevisiae (yeast). This test was intended to be a positive control to our planned system and was conducted by applying several specified concentrations of the fungicides into both agar and suspension growth medium of the organisms and prove the fungicide effects on their growth rate. Subsequently, this test should identify the most effective fungicide concentration and determine the work principles and side-effects of fungicides such as lethal and growth-suppressing effects. Moreover, we also attempted to “evolve” our model organism S. cerevisiae by determining the organism’s minimum growth-effective fungicide concentration and inoculate them into the same suspension medium with increasing concentration each time to prove the formation of quantitative resistance. We also tried to compare the effects of the used fungicides to other organisms such as our lab-grown wheat seedlings.
The starting point to the fungicide test is to determine the appropriate amount of fungicide to be used within laboratory scales.
The fungicides Folicur® and Proline® were delivered to our laboratory along with the safety data sheet, from which the recommended working
concentration of each fungicide is assessed. Since the given data are orientated for the fungicide usage in the agriculture field,
we ought to convert the units from the agriculture field to the ones from agar plates or suspension cultures in the laboratory.
The conversions are as follows:
The recommended dose of Proline is 0.8 l/ha in 200 – 400 l/ha water. Meanwhile for Folicur is 1.5 l/ha in 200 – 400 l/ha water. Hereby, we chose to solve the fungicide in the maximal amount of water (400 l/ha). Given that the surface area of an agar plate is 58 cm2, we converted the needed amount of fungicide in an agriculture field to the needed amount in agar plate, which are listed in the table below:
Conversion of fungicide units in an agriculture field and fungicide units on an agar plate for the growth experiment of S. cerevisiae and A. niger.
Fungicide
Field Usage Dose
Plate Usage Dose
Volume for Every Agar Plate
For Every 10x Fungicide Solution (in 21 ml water)
Proline
0.8 l/ha in 200-400 l water/ha
4.64 μl in 2.32 ml water
2.32 ml
420 μl
Folicur
1.5 l/ha in 200-400 l water/ha
8.7 μl in 2.32 ml water
2.32 ml
787.5 μl
The next step is to solve the fungicide stock solutions to the 10x amount of the required usage dose with distilled water according to the table. From this 10x solution, we performed a serial dilution to 1x, 1:10, 1:100, and 1:000. The diluted solutions are subsequently added to the suspension and agar growth mediums in the volume of 2.32 ml for each 30 ml of medium.
After the growth mediums are composed and the fungicides are diluted, the model of the experiment should look like this:
Experiment plan for the fungicide simulation in both suspension and agar growth medium. Fungicides are diluted according to the diagram and added to the growth medium.
Basically, every growth medium (suspension and agar) would consist of 30 ml medium and 2.32 ml of the fungicide concentration of choice. An overnight culture of the respective model organism is to be inoculated a day or two days prior to the experiment. The growth experiment of the two model organisms would be carried out separately to avoid contamination and the time span of the experiment is to be adjusted with the organism. For example, A. niger would need more time to grow and thus, the experiment time should be extended accordingly. The agar plates are to be incubated at 30 °C for several days until the growth spurs are visually identifiable.
Saccharomyces cerevisiae
For the experiment series with Saccharomyces cerevisiae (yeast), we prepared the YPD (Yeast Peptone Dextrose) medium following the respective standard protocols and added 15 g of agar for the preparation of agar medium for YPD agar plates.
We carried out the first step of the experiment plan by inoculating an overnight culture of S. cerevisiae in YPD-medium (suspension) from an agar plate and incubate at 30 °C and 180 rpm on a shaker. For the growth experiment, the OD600 of the overnight culture is measured on the following day to determine the volume of the overnight culture to be inoculated in a fresh medium. Followingly, the start OD from all the culture medium is adjusted to 0.1 by adding the right amount of volume from the overnight culture to the new culture according to this formula:
To ensure the authenticity of the comparation, we composed a negative control containing YPD medium without any fungicide and the S. cerevisiae cells from the overnight culture. The flasks were all incubated in the same shaker at 180 rpm and 30 °C. The OD600 of each culture is to be measured every hour for 6 hours long. Additionally, we distributed 100 μl of the overnight culture to YPD agar plates with the same amount of fungicide to observe for growth.
Aspergillus niger
The similar cultivation and measurement methods are then carried out for Aspergillus niger. We also started by inoculating an overnight culture of Aspergillus niger to the CM growth medium and inoculate the overnight culture to CM suspension medium with each different fungicide concentrations and one without fungicide as a negative control. The cultivation lasted for 12 hours and the OD measurement took place every 3 hours. Additionally, 1 ml sample of the culture was taken from each point of the measurement to determine the dry biomass. This serves as a second control point for this growth experiment due to the organism’s filamentous nature, which could interfere with the OD measurement. For the preparation of the dry biomass samples, the required amount of microcentrifuge tubes is incubated overnight at 60 °C until dry. The weights of the dried tubes are to be measured and documented. At the end of the cultivation, each of the taken samples should be transferred separately to the dried microcentrifuge tubes. Centrifuge the samples accordingly and discard the supernatant. Incubate the remaining sample inside the tube at 60 °C until dry and measure again the new weight of the microcentrifuge tubes. Finally, the dry biomass is the weight difference between the tube with the sample and the tube without sample.
The growth experiment also took place in the CM agar plates with different concentrations on fungicide.
Resistance Test
Resistance test against fungicide of the model organism in their respective suspension growth medium.
Due to their relatively rapid growth compared to A. niger, we decided to additionally carry out a resistance test for S. cerevisiae. After the last concentration in which the organism still survive is determined, we used this concentration to start the resistance test. For this, we inoculated an overnight culture of the organism in the respective growth medium and started cultivation after exactly 24 hours with the highest fungicide concentration in which the organisms still withstood the conditions during the growth experiments. A start OD of 0.1 was aimed and to be documented. Every 12 hours, we measured the OD to test for growth and every 24 hours, we inoculated the previously analyzed culture to a fresh growth medium with higher fungicide concentration and the same fungicide concentration as a negative control. This test goes further to see the mechanism of the quantitative resistance formation.
Fungicide Treatment on Lab-grown Wheat Seedlings
Dropping fungicide liquid onto the agar plate with the infected wheat seedlings.
Our lab-grown wheat seedlings inside agar plates exhibit signs of fungal infection. We used this opportunity to do a fungicide treatment using both Folicur and Proline. For this, two plates containing the infected wheat seedlings are selected randomly and some pictures before the treatment were taken. One plate will undergo the treatment with Folicur and the other one with Proline.
Identical to the other experiments, 2.32 ml of the fungicide were dropped to the agar plates containing the wheat seedlings within the recommended concentration. The progress is to be documented within the next several days to see signs of healing.
Results and Discussions
In this section, the results of the experiment series on S. cerevisiae and A. niger are to be documented separately. The entirety of the conclusions from both organisms are to be compared and summarized.
Saccharomyces cerevisiae
The growth experiment of S. cerevisiae during 6 hours of cultivation in YPD medium with different fungicide concentrations resulted in the following growth rate:
Growth rate of S. cerevisiae with Proline (concentrations 10x – 1:1000) and Folicur (concentrations 10x – 1:1000) each with a negative control (-) which contained no fungicide in the medium.
From both diagrams, it could be concluded that even the least amount of fungicide (1:1000) would already interfere with the growth speed of S. cerevisiae compared to the negative control (-). Meanwhile, increased amounts of the fungicide (1:100 and 1:10) would slow down the growth respectively, but not enough to stop it ultimately. It is also interesting that starting from the recommended amount (1x), no growth was detectable, suggesting that the recommended amount and beyond have indeed lethal effects to the growth of S. cerevisiae.
Apart from the similar tendencies that both fungicides exhibited to this organism, it is still recognizable that Folicur might be more effective than Proline, given that cultivation with Proline during 6 hours ended with a higher OD than the cultivation with Folicur (see fig. 6).
After a week of incubation at 30 °C, the YPD agar plates were also analyzed.
YPD agar plates containing plated 100 μl S. cerevisiae overnight culture with both fungicides at different concentrations (10x – 1:1000) and without fungicide at all as negative control (-) on the left side.
From the visually identifiable growth on agar plates, it can also be concluded that the recommended amount (1x) and beyond affected the growth of S. cerevisiae in a lethal way and that Proline might really be less effective than Folicur. At 1:10, both YPD Proline and YPD Folicur plates still exhibit growth, although not visible enough to be caught on camera. At decreasing rate of fungicide (1:100 and 1:1000), faint growth spurs are also visible on the YPD Folicur plates. Meanwhile, the YPD plates with Proline showed an essentially clearer growth spurs at the same fungicide amount, which furthermore supports our statement about Proline’s relatively lower activity compared to Folicur.
In accordance with the results, we started the resistance test from the concentration of 1:10 and went up further to 1:8, 1:4, 1:2, and finally 1x. After 5 days of measurement every 12 hours and inoculating into a medium with increased fungicide concentration further every 24 hours, we came up with the following result:
The resulted quantitative resistance formation against Folicur from S. cerevisiae. The plotted OD values for each day and each concentration are based on the ODs from the start and the end of each cultivation.
The resulted quantitative resistance formation against Proline from S. cerevisiae. The plotted OD values for each day and each concentration are based on the ODs from the start and the end of each cultivation.
Through both diagrams, the formation of quantitative resistance against both fungicides are made visible and comparable. Despite decreasing growth rate with the increasing concentration, the “evolved” S. cerevisiae cells are proved to be able to withstand the lethal concentration (1x) of both fungicides at the end of the experiment. Additionally, Proline still exhibited a more rapid growth rate at the higher concentrations, which once again could correlate to Proline’s less efficiency relative to Folicur.
Aspergillus niger
Compared to S. cerevisiae, the whole growth experiment with A. niger was more complicated due to the organism’s characteristics and slow growth rate, which interfered with the OD measurement.
Despite the circumstances, we were able to detect the growth through measurement of dry biomass.
Measured dry biomass of A. niger during 12 hours of cultivation. The start weights are standardized to 0.1 mg.
Like S. cerevisiae, A. niger also showed no signs of growth starting from the 1x concentration. Furthermore, the cells also exhibited a higher growth rate with Proline than with Folicur. These results finally supported the claims from the S. cerevisiae results. After some days, the growth rate on the agar plates were also evaluated.
CM agar plates containing plated 100 μl A. niger overnight culture (cultivated for about three nights) with both fungicides at different concentrations (10x – 1:1000) and without fungicide at all as negative control (-) on the left side.
From the growth of A. niger on the agar plates, growth spurs are also identifiable up until the fungicide concentration of 1:10 before disappearing completely on the agar plates with 1x concentration and above. It is also noticeable that the Proline plate showed more advanced growth than the Folicur plate. However, we observed an unusual tendency at the very faint growth spurs on the plates with 1:100 and 1:1000 fungicide concentration. One of the possible explanations for this inconsistency is the uneven dispersion of the A. niger overnight culture, which might have contributed to the lack of the cells plated on the respective agar plates.
During these experiments, we also stumbled upon an interesting result which was based on the contamination on our first batch of A. niger agar plates. Before starting over, we took the time to observe the contaminants briefly and came to the assumption that the contamination might originate from colonies of unidentifiable bacteria due to the rapid growth (incubation time less than 2 days).
Contaminated CM agar plates.
Interestingly, this contamination also stopped at the concentrations of 1x and 10x, which may suggest that the fungicide does not only stop at disrupting fungal growth activities, but also the growth of other organisms that happen to be around, including bacterial contaminants.
From these series of experiments, we could conclude that the recommended usage dose from Bayer AG is appropriate and sufficient to eliminate pathogens in the field. Therefore, it shouldn’t be necessary to increase this concentration and it might even be reasonable to determine the lowest effective concentration before applying the fungicide to avoid using higher concentrations unnecessarily.
On the other side, Folicur has been showing signs of superior ability to eliminate pathogens compared to Proline throughout the experiment. This superiority might be due to higher concentration aimed by the safety data sheet (Proline: 0.8 l/ha in 200 – 400 l/ha water. Folicur: 1.5 l/ha in 200 – 400 l/ha water). Furthermore, Folicur also exhibits more hazard warnings and therefore proved itself to be more toxic than Proline, which explains the increased effectivity in eliminating pathogens.
Comparation of Folicur’s hazard warnings (right) to Proline’s (left).
In this case, it might be essential to plan a fungicide treatment on crops thoroughly. Apart from the severity of the infection, the effect of the fungicide use on the surrounding environment should also be included into consideration.
About a week after we treated our infected wheat seedlings with the fungicides, we documented the following photo of the plates:
Wheat seedlings a week after the fungicide treatment.
From these results, it could be concluded that the fungal infections were eliminated not in the sense of removing the fungal bodies from the plant, but rather to stop the infection from spreading. This result correlated to the growth experiments with the model organisms, in which the OD or the dry biomass stayed in a constant value as a sign of disrupted growth rate. The comparison for this experiment should work even better when using wheat seedlings that are still on the first stages of the infection and using a negative control with untreated seedlings.
Overall, it is also worth mentioning that the whole experiment took place within laboratory scales (agar plates and cultivations in 30 ml medium) with relatively simple model organisms, which obviously contributed to the rapid progress and the possibilities to obtain results in a relatively short time. On the scale of an agriculture field, all these progress would take a significantly longer time, also considering that fungal pathogens in the field are morphologically and physiologically more complex than S. cerevisiae or A. niger. Nevertheless, this experiment could serve as another perspective to the utilization of fungicide and the danger of quantitative resistance.
Bayer logo.
For the last part, we would like to thank Bayer AG for giving us the opportunity to carry out these series of experiments. It has been a fascinating experience for us to test the provided fungicides in our laboratory.
Further possible applications of Troygenics
Since we could proof our concept in the non-pathogenic model organisms S. cerevisiae and A. niger, our Troygenics can be
adapted to fight several plant pathogenic fungi. Numerous experts pointed out that pathogens like for example Phytophtera
infestans, Puccinia graminis and Fusarium oxysporum pose a huge threat to the world’s food supply.
But apart from fighting plant pathogenic fungi and simplification of lab work regarding fungi and other eukaryotes difficult
to transform, there is a variety of possible applications for our Troygenics. Starting from fungi, not only plant
pathogenic fungi pose a dangerous threat. Fungi, that infect animals and even humans constitute underestimated dangers.
There are already bat-or toad-infecting fungi that endanger whole species which would have a terrifying impact on entire
eco systems. To adapt our Troygenics to threats like those, tere are only small modifications possible. A target specific
ligand has to be fused to the major coat protein pVIII and short target specific guideRNAs have to be implemented into the
CeDIS.
But fungi are not the only eukaryotic pathogens. Trypanosoma for example, that cause the african sleeping sickness and
often result in the patients death, are very difficult to target, too (WHO). Since our Troygenics would specifically
fight the Trypanosoma while having no effect on the human cells, they show great advantages to conventional
treatments. Usually, Trypanosoma are treated with chemicals. Those chemicals have to cross the blood-brain-barrier,
like Trypanosoma do, too. Unfortunately they can show severe side-effects that result in serious brain-damage (WHO).
As a conclusion, the developed platform system Troygenics shows great potential to overcome unsolved problems,
that are currently on the rise, in a variety of different fields.
