PROOF OF CONCEPT

INTRODUCTION

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EXPERIMENTAL DESIGN

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  The experimental steps are described on the Experiments page. All the necessary safety precautions were taken, and are described on the Safety page.

  Our experimental design consisted in these steps:

• Transforming Dickeya solani with four different plasmids: pSEVA521 (empty vector), pSEVA521-fiatlux, pSEVA531 (empty vector) and pSEVA531-fiatlux. The two plasmids pSEVA521 (see Material) and pSEVA531 (see Material) are two mobilisable vectors with RK2 and pBBR origins of replication respectively and carry a tetracycline resistance gene. We wanted to check both backbones. We also checked if the bacteria were luminescent when transformed with the plasmids containing the fiatlux operon: this was the case (Figure 1)!

• Studying the effect of antibiotic concentration on emission of luminescence. We concluded that luminescence of D.solani (with pSEVA521-fiatlux and pSEVA531-fiatlux) is much stronger on Petri dishes with a higher concentration of tetracycline (Figure 1) and the higher the concentration, the later the maximum of luminescence is reached. We also verified this in a liquid culture.

Figure 1 - Testing luminescence in D. solani with the following plasmids: pSEVA531-fiatlux, pSEVA521-fiatlux, pSEVA531, pSEVA521. Different tetracycline concentrations were tested (0, 3, 5 and 10 µg/mL), after 24h and 48h. Images were taken with a high-sensitivity camera. Luminescence can be observed for the strains containing plasmids with the fiatlux operon.

• Studying the effect of the operon on the bacteria’s growth (energy consuming, etc). For this we compared the bacterial growth, by measuring the optical density OD, in several conditions: bacteria transformed with empty plasmid pSEVA521 or pSEVA531 and bacteria transformed with both plasmids containing our fiatlux operon (effect of operon expression on growth). We then modeled the bacterial growth by plotting the OD as a function of time: we saw that the bacterial growth was not significantly impaired by fiatlux (Figure 2).

Figure 2 - Optical density of the cultures of Dickeya solani with the 4 different plasmids (pSEVA521, pSEVA531, pSEVA521fiatlux and pSEVA531fiatlux) as a function of time

• Studying the stability of the FIAT LUX plasmid in Dickeya solani. For this, we developed a specific protocol (see Plasmid stability test on the Experiments page ). In the absence of a selective pressure (the antibiotic), the plasmid might get lost in the generations to come. This control is essential for iGEM teams wishing to ensure that the plasmid remains stable in their bacteria. We concluded that the plasmid was stable in Dickeya solani for at least 27 generations (Figure 3). This is good news, as it means that it allows a long-term in vivo study on plants with bioluminescent Dickeya solani, in order to test a treatment for example.

Figure 3 - Stability test results: empty pSEVA531 versus pSEVA531-fiatlux transformed D.solani. Number of bacteria was accounted for over a 3 day period, and 2 replicates per plasmid were performed. “LB LB” corresponds to bacteria that were first grown on an LB medium and then re-isolated on an LB medium, thus corresponding to the total number of bacteria, “LB Tet” are bacteria that were first grown on an LB medium and then re-isolated on a Tet medium, thus corresponding to the bacteria containing our plasmid, “Tet Tet” are bacteria that were first grown on a Tet medium and then re-isolated on a Tet medium, thus corresponding to a global bacterial growth control. “LB LB” and “LB Tet” number of bacteria are compared in order to define if the plasmid is well kept by D.solani bacteria.

• Infection of plants. The last step before validating the proof of concept consisted in infecting plants, and checking if the luminescence could still be detected. We infected chicory leaves and potato tubers with Dickeya solani containing the following plasmids separately: pSEVA521-fiatlux, pSEVA531-fiatlux, as well as pSEVA521 and pSEVA531. The idea was to observe luminescence during an infection by Dickeya solani. Thanks to our software, we analyzed the recorded images and checked that Dickeya solani produced sufficient luminescence to track the path of bacteria.

  We were able to successfully observe luminescence during the development of infection on chicory leaves (Figure 4) and potatoes (Figure 5)! We also demonstrated that the plasmid pSEVA531-fiatlux was more efficient than pSEVA521-fiatlux regarding produced luminescence. As a result, we could also see that the plasmid did not affect the bacterial development in the plants and the infection dynamic.

  Even so, we were extremely proud to see that our proof of concept was successful! Indeed, we were able to obtain bioluminescent pathogenic bacteria (Dickeya solani), and successfully infect plants, allowing its in situ study.

Figure 4 - Timelapse of the infection of our bioluminescent Dickeya solani on chicory leaves

EXTENSION OF THE USE OF FIAT LUX TO OTHER PATHOGENIC BACTERIA

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  We also wanted to extend the proof of concept to as many bacteria as possible. We tested the two following bacteria:

  Again, the necessary safety measures were taken, and they were handled in an academic laboratory classified level 1 of biosafety (standard microbiological lab). These strains were transformed with pSEVA521, pSEVA531, pSEVA521-fiatlux and pSEVA531-fiatlux by conjugation, as with Dickeya solani. Luminescence was observed for these two strains (Figure 6)!

CONCLUSION

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REFERENCES

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