Team:Toulouse/Result/experimental-results

We used and developed several protocols to demonstrate the existence of chemotaxis in B. subtilis wild type (WT) strain and SubtiTree bacterium towards N-Acetylglucosamine.

1. Petri Dishes Test

We first wanted to visualize chemotaxis on Petri dishes. We hoped to obtain pictures with bacteria halos directed or around attractive components and thus tried different protocols. The first protocol was adapted from the one published by the Imperial College 2011 iGEM team. They put the attractive compound on a paper disk in the middle of a Petri dish containing a medium with 0.3% agar. Cells are loaded in this medium (Figure 1).

We first tried to test chemotaxis onto Petri Dishes filled with a 0.3% agar medium. This semi-solid medium allows the bacterial motility. A paper disk containing an attractive compound is placed in the middle of the dish and cells are then loaded in the medium (see Figure 1). This protocol was taken from the the Imperial College 2011 iGEM team.

Figure 1: Scheme showing how cells are filled into the medium. (A) A pipet tip is used to deposit cells in the gelose. (B) Bacteria should move toward the attractive compound which diffuses.

We did not have any result with WT Bacillus subtilis and glucose as attractive compound (Figure 2-A). B. subtilis is attracted by many other glucides and amino-acids, so we also tried to test diluted glucose in LB medium attractant (Figure 2-B).

Figure 2: Chemotaxis test with Glucose as attractive compound (A) and Glucose added to LB medium as attractant (B).

We could not notice any difference between the petri dish with or without glucose. With an addition of LB medium to sugar, a large halo around the paper disk was noticeable. This halo may correspond to cells attracted by the solution, as it is not noticeable when cells are not added (data not shown). Anyway we did not have enough reproducible and reliable results to be satisfied with this test.
Furthermore, with the addition of LB medium, it is hard to make the distinction between the attractive effects and the simple growth resulting from random diffusion.
We have started new tries using different protocols.

2. Plug in Pond system

This protocol we worked on is taken from a thesis (see references [1]). . A solution of B.subtilis is grown overnight so as to obtain a cell density of 8x10⁸ cells/mL. 10mL of the solution is mixed with 15mL of LB medium with 1.5 % agar kept at 45°.The final concentration of the obtained medium is 0.9% agar. Tetracycline is aded at 25µg/mL, in order to inhibit growth and to only observe the chemotaxis phenomenon. Plates are cooled and dried, before digging wells with a punch or 1mL tips. The wells are filled with attractive compounds (Figure 3). After one hour at room temperature, photos of the plates are taken and the results are analyzed.

Figure 3: Schema showing how are made plug-in-pond tests.

On our first try with B. subtilis, we made three wells per plate (Figure 4).The wells were filled with glucose at different concentrations and tetracycline was not added in one of the plates.

Figure 4: Plates after 12h at room temperature.

After an hour, no tangible results were obtained. It is only after 12hours that we were able to observe halos around the wells with glucose at 1M in the plates without tetracycline. Tetracycline concentration seems to be too high and inhibits any bacterial activity. Therfore, we have worked with tetracycline at 15µg/mL. We tried this protocol again with this new condition. We made two wells per plate (Figure 5), one with either Glucose or N-acetyl-glucosamine and one with LB medium. As previsously, no results were achieved after 1h, but after 12hours we could notice halos.

Figure 5: Chemotaxis test with Bacillus subtilis WT. The upper wells contain attractive compound and the lower contain medium without attractive compound.

Results are not as clear as the first time, but we observed halos around the well with glucose at 250mM with and without tetracycline. We have then tried the same experiment with N-acetyl-glucosamine and we did not see any halo in the tested conditions. Thus we assumed that our B. subtilis 168 strain was not attracted to N-acetyl-glucosamine. However, the results are not clear, reliable and reproducible enough with the plug-in-pond protocol. Another testing protocol was then adopted.

References:
[1] : Etude de la réponse adaptative à l'oxyde de triméthylamine et de son mécanisme de détection chez Escherichia coli et Shewanella oneidensis, 2008, Claudine Baraquet, université de la méditerranée Aix-Marseille II

4. Capillary test between two tubes also called the tubes test

After the experiment of the plug-in-pond, we decided to construct a system by welding two Eppendorf tubes with a capillary thanks to an electric burner.

Figure 6: Photography of the first tubes system

We tested this system with a fuchsin dye and water and we were able to observe the diffusion of fuchsin towards water. However this construction had a leakage next to the weld seam that we could not stop. Thus, we asked the help from the INSA glass blower, Patrick Chekroun. He designed two systems composed of two tubes linked by a capillary.

Figure 7: Scheme of the tubes system

As we did previously, we tested this new system with fuchsin. This experiment was made with WT Bacillus subtilis and N-Acetylglucosamine.

NB: We could not see the diffusion from one tube to the other. We made the hypothesis that it was not visible by sight because of the small diameter of the capillary.

The following strategy was used to avoid disturbance due to pressure and liquid movement through the capillary:
- The first step was the addition of Wash Buffer until the capillary was full to avoid the presence of air bubbles which could lead to diffusion problems.
- Then, the tube 2 was plugged with the thumb while another person was adding the bacterial solution of WT Bacillus subtilis in the tube 1.
- The tube 1 was also plugged and only after the thumb could be removed from the tube 2.
- In the same way, the N-Acetylglucosamine was added in the tube 2.
- The same process was made with a xylose positive control.

NB: According to the article Chemotaxis towards sugars by Bacillus subtilis, (George W. Ordal et al., 1979), glucose and xylose have the same attractant power. We prefer a positive control instead of a negative one because we were not sure that this system was efficient.

- The system was kept straight for 2hours. Every 40 minutes, we took a sample of each tube and spread it on an agar plate (dilution 1/1,000).

Figure 8: Photography of the tubes system

Unfortunately, the dilution was too high to detect any chemotaxis movement and the time was too short. We did not find any information in the literature.
As we did not have the time to optimize this protocol we preferred using the protocol of the 2011 Imperial college iGEM team : the tips capillary test.

5. Tips capillary system

First tips capillary system

This protocol comes from 2011 Imperial College iGEM team and was adapted by our team in several steps (See chemotaxis protocol).
In the first tips capillary system, we used parafilm to avoid any kind of air disturbance in the tips. The different steps are described below:
- 15µL of each chemo-attractant was pipetted.
- The bottom of tip with the pipette was then put on a piece of parafilm and the pipette was removed from the top of the tip.
- The top of the tip was then sealed with a piece of parafilm. By this way, the sterility can be assured and the liquid stays inside the tip.
- To finish, the level of the solution in the tip was marked.

Figure 9: Sealing of a tip with parafilm

- After all the attractants were added in the tips, we put them on a green base to carry them. The whole process can be seen on Figure 10.
- Each tip was immersed in 300 µL of a bacterial solution in the wells of an Elisa plate.

Figure 10: First tips capillary system

NB: the yellow carton was used to stabilize the system and keep it straight.

- After one hour, the tips were removed from the bacteria solutions and the content of the tips was observed with a Thoma cell under the microscope.

We had several problems with this system:
- The liquid level decreased during the experiment and we did not have enough liquid to fill the Thoma cell. Thus, it was not possible to count.
- The bacteria were moving and therefore, we could not proceed to a bacteria count.

Regarding these observations we decided to spread the tips content on agar plates instead of using Thoma cell and microscopy.

Second tips capillary system

Figure 11: Second tips capillary system

Improvement of the second tips capillary system

However this system was not optimal it is why we decided to use blu tack instead of parafilm:

Figure 12: Improvement of the second tips capillary system

At that point, the protocol was approved and the final test could finally start! :-)

There was just one tiny problem… we did not have our optimized bacterium transformed with the chemotaxis module!!! That is why we concentrated our efforts on WT Bacillus subtilis strain.

The main goal was to find an optimized control and to analyze the eventual chemotaxis of the WT strain. To avoid osmolality bias, we wanted to find a molecule which was non-attractant and with a similar molecular weight than the N-Acetylglucosamine (221.21 g/mol). Our first idea was to use fuchsin (Molecular weight: 337.85 g/mol).

At the beginning, the experiment was conducted with only one negative contraol, the fuchsin and different NAG concentrations: 25mM, 250mM and 500mM. The tested strain was Bacillus subtilis 168:

Figure 13: Fuchsin - negative control (dilution 1/50)	Figure 14: NAG (25mM) (dilution 1/50)

The average number of colonies with the negative control is 121. On the contrary, a cell layer is observed for the NAG plates with every concentration.

Thus, we assumed that WT Bacillus subtilis was more attracted by NAG than fuchsin. Indeed we can neglect the bacterial growth because the test only lasts one hour. We also neglect diffusion and osmolality phenomena for the previous reasons.

Unfortunately for us we forgot one major effect… Can you believe that fuchsin solution contains about 15% of ethanol?!!! This concentration can lead to the death of some cells which probably happened to our results.

This incredible and dramatic discovery destroyed all of our hopes about the God of chemotaxis! :-(

However, our team did not give up on synthetic biology ! :-) Indeed, after days of disappointment and no time left for lab work, we raised from ashes and tried to find another negative control.

Hopefully, we managed to find a negative control: galactose (25mM). The article Chemotaxis towards sugars by Bacillus subtilis (George W. Ordal et al., 1979) proved that it was a poor attractant.

We made our tests again with this new molecule and glucose (25mM) as positive control.

Figure 15: Final results (dilution : 1/10,000)

The miracle arrived! We managed to prove that our WT Bacillus subtilis was indeed naturally attracted to NAG.

NB: It was our last experiment. Unfortunately we were running out of time and we could not do much more test. We would like to do the experiment with a lower dilution and repeat it several times.

Our results are not statistically significant however this result has been proved in literature.

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1. Preliminary experiments

Purpose

The first experiment deals with the culture conditions to see if Bacillus subtilis can resist to a low temperature and with the CBB buffer. To do that, several bacterial concentrations have been tested starting with an OD of 0.1 and diluting this solution to get estimated ODs of 0.05, 0.025, 0.01. These different Bacillus subtilis solutions were incubated 1 hour at 4°C with 500µL of CBB or water. Finally a cell count on Thoma cell counting chamber was performed.

Results

The bacterial solutions could not be counted because of two major problems: the too high number of bacteria with the 0.1 OD or the too low number of bacteria with the 0.01 OD. Thus, the study is mostly focused on the intermediate values (Figure 16).
First of all, a same cell concentration can be noticed with the presence of CBB or water with estimated ODs of 0.05 or 0.025. Moreover, twice less cells can be found in the lowest concentrations in bacteria comparing to the 0.05 OD concentration which agrees with the dilution ratio.
Thus, the experimental conditions regarding the presence of CBB and the incubation temperature at 4°C do not harm the cell surviving.

Figure 16: CBB presence has no effect on bacteria. The bacterial concentration was measured regarding the presence or the absence of CBB for the observed OD (0.1) or estimated ODs (0.05, 0.025, 0.01).

2. Binding test using engineered B. subtilis

Purpose

Transformed Bacillus subtilis with the binding module is able to produce a protein composed of the bacterial peptidoglycan bonding of LycT and the GbpA 4th domain of Vibrio cholerae allowing the chitin bonding. The synthetic bacterium is put with special beads composed of the polymer miming the fungal pathogen wall. After several washes, bacteria specifically attached to the chitin are put on plates and counted.

Results

The first observation is that both bacterial solutions of wild type Bacillus subtilis and SubtiTree have the same concentration : 105 bacteria/mL (Figure 17). Even though there is no significant difference between both strains after the first wash, the second wash has a major effect since it allows 40 times more Wild Type bacteria to come off the beads. This result correlates with the number of bacteria binded to the beads for the synthetic strain with the binding module.
Thus, the binding system seems to function correctly and leads to the bacterial attachment on the chitin.

Figure 17: Attachment of Bacillus subtilis with binding module to chitin. The WT bacteria or the bacteria with the binding system concentration has been determined during the different steps of the binding test. The stars represent a significant difference observed with a Student test with p<0.05.

3. Microscopic observations

Purpose

We want to observe the SubtiTree's binding on beads coated with chitin. In order to perform a 3D reconstruction showing this interaction, we use confocal laser scanning microscope. Through the use of a fluorochrome (Syto9), we can highlight the presence of bacteria on the surface of the beads (individualized by phase-contrast). A first calibration step determined the minimum threshold to remove the background noise and the natural fluorescence.

Results

First, we note the great bacterial presence on the surface of beads coated with chitin. These images seem to highlight their interactions.

Figure 18: Microscopic view of beads surfaces coated with chitin

Using ImageJ software, we are able to create 3D pictures and movies of those comments.

Figure 19: A short movie of 3D bead surfaces coated with chitin

Finally we want to observe the bacteria after the second wash. When our bacterium has the binding module, results suggest a lower number of bacteria in the washing solution. SubtiTree is retained by the beads.

Figure 20: Microscopic view of bacteria after washing

Finally, overall results are consistent with the presence of functional binding system.

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1. Preliminary experiments

Tests with commercial peptides and controls

The first tests were accomplished with commercial GAFP-1 and D4E1 peptides at different concentrations (12.5µM, 25µM, 100µM). These tests were performed on different fungal strains sharing the same phylum with Ceratocystis Platani. As Ceratocystis Platani is pathogenic, we could not perform tests directly with this fungus.
After several days at 30°C, the PDA (Potato Dextrose Agar) plates covered with fungus and commercial peptides were analyzed.

An inhibition halo was noticeable with commercial D4E1 peptide at 100µM on Aspergillus brasiliensis. Less bright halos were also present with lower concentrations. Concerning commercial GAFP-1, we did not notice any effect in the tested conditions. As positive control, a well-known chemical fungicide was used: the Copper Sulfate. The inhibition of the fungal growth was complete at 20mg/ml, and at 10mg/ml a darker halo appeared around the pad filled with Copper Sulfate as we can see on the figure below. This corresponds to a sporulating halo in response to the stress generated by the fungicide.

Figure 21: Results of the preliminary tests

Regarding these results, we concluded that very high fungicide concentrations are required to inhibit the fungal growth. Following these tests, new conditions were adopted in order not to encourage too much fungal growth over bacterial growth. The culture medium was adjusted to fit our objective and to approximate the conditions found in the trees: a 'sap-like' medium was elaborated. The incubations were then carried at room temperature.

2. Test with SubtiTree

In order to test Bacillus subtilis mutants, it was essential to find the right balance between the fungal growth and the bacterial one. This condition was necessary to get a high concentration of peptides. In our genetic constructions, these peptides are designed to be exported in the extracellular medium.

The transformed Bacillus subtilis strains grew at 37°C during 72h and were tested. After centrifugation, the supernatant and the resuspended pellet were placed on pads and disposed on plates previously seeded with a defined number of conidia (see protocols to have more details). After several days at room temperature, an inhibition halo of Trichoderma reesei's growth was clearly observable for the strain expressing D4E1 gene. The inhibition was even more noticeable with the strain carrying the GAFP-1 + D4E1 operon (see the photos below).
However, no effect was detected for the strain expressing the GAFP-1 gene, supposing a synergistic effect between these two peptides.
Regarding EcAMP and the triple-fungicides operon, no effect has been detected on the fungal growth. Several factors can explain these results: a number of post-transcriptional modifications are required to have a functional EcAMP and in addition to that, sequencing results of these constructs showed some differences with the original designed sequence.

Inhibition halos are not visible with supernatants, probably because of their low concentrations in the extracellular medium. Another effect was noted with the same strains expressing D4E1 and GAFP-1 + D4E1 on another fungus Aspergillus brasiliansis. This effect is comparable to the one previously noted with a low concentration of sulfate copper.

Figure 22: Results with transformed bacteria.

The choice of our chassis appears to be optimal as we noted that wild type Bacillus subtilis disturbs the hyphae growth of the fungi. Some strains of Bacillus subtilis (qst 713) are already used as Biofungicides for use on several minor crops to treat a variety of plant diseases and fungal pathogens.
After this set of experiments, the strains expressing D4E1 and expressing GAFP-1 + D4E1 have shown to be the best candidates to play a major role in the fight against fungal diseases such as Canker stain. Keeping in mind our objective, we decided to tests these strains in model plants: Nicotiana benthamiana and Arabidopsis thaliana.
These tests were performed in the National Institute for the Agronomic Research by experts in this domain.

3. In planta tests with SubtiTree

Figure 23: Injection of SubtiTree in a model plant

The goal of the project is to introduce the transformed bacteria in a diseased tree. So it is necessary to perform in planta tests to judge the fungus-killing abilities of the two strains selected after the previous set of experiments.
SubtiTree is first inoculated in two model plants (Arabidopsis thaliana and Nicotiana benthamiana). After this step, a phytopathogenic fungus (Sclerotinia sclerotiorum) is placed on the leaves.
These tests were made in association with Sylvain Raffaële and Marielle Barascud of the National Institute for the Agronomic Research laboratory.

Twenty-four hours after SubtiTree inoculation, no phenotypic modification of the leaves can be detected. We can conclude that our bacterium, its introduction and the fungicides production in plants do not have deleterious effects.
Without proper treatment, the drop of the pyhtopathogenic fungus on Nicotiana benthamiana's leaves causes a necrosis halo which can be measured after 40h. The lesion size and the number of inoculated sites seem to be reduced by B. subtilis expressing DE41 or GAFP1-D4E1, unlike with the WT bacterium. A second set of experiments is expected to be more statistically precise.


We did not observe any significant results for Arabidopsis thaliana because of the use of two plants batches with different ages.
We can therefore conclude that when SubtiTree is in plant's physiological conditions, it is harmless to the plant, and that the production of fungicides is effective, reducing the leaves necrosis.

Figure 24: Results of in planta test

Thanks to the diversity of anti-fungal peptides, this strategy can be adapted to different types of diseases, with different degree of specifity, etc.

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