Logo fiat lux




  After engineering our parts in E coli, we concluded that our FIAT LUX operon was functional, as we were able to observe luminescence. We also characterized the tool in E coli. However, we kept in mind our primary goal, which was to use FIAT LUX to track pathogenic bacteria in situ. To validate our tool, we had to transfer our construction into pathogenic bacteria, check that luminescence was still produced, and then successfully infect plants, to validate our proof of concept and show that it worked in the real world.

  We wanted to do our proof of concept on a real-life problem. We chose to work with Dickeya solani, for several reasons:

  • This bacteria had already been thoroughly studied by the MAP laboratory, where our PIs work and where we did our experiments, so this was easier for our team.

  • D.solani can be cultivated in a lab, which was a necessary criterion for our proof of concept. However, after doing research, we found that the bacteria responsible for the most severe crop devastations can be cultivated (Mansfield et al. 2012).

  • Also, handling D.solani didn’t require more than an academic laboratory classified level 1 of biosafety (standard microbiological lab). Also, this is not a known pathogen for humans.

  Finally, this bacteria is responsible for causing the blackleg disease on potato tubers. This emerging strain is a global problem. Indeed, potatoes are the target of many diseases (AHDB 2022), such as late blight, silver scurf, or dry rot. For more than 15 years, a new crop pathogen has been emerging: Dickeya solani (Toth et al. 2011). Potatoes being one of the most consumed foods in the world, the consequences of diseases affecting these crops are of great concern. Dickeya solani is responsible for causing the blackleg disease of tubers, characterized by the appearance of soft rot, in temperate zones (Toth et al. 2011). Since the appearance of Dickeya solani, the losses caused have been increasing significantly, and the contaminated areas are spreading all over Europe, even in Israel (DAERA 2017). These agricultural diseases can cause tremendous losses: in the Netherlands, Dickeya caused more than 30 millions of euros in economic losses in 2011, though this number is nowadays largely underestimated, as not much is known about Dickeya’s propagation dynamic (Toth et al. 2011, Dupuis et al. 2021).

  There is currently no effective treatment for the blackleg disease caused by Dickeya. The only solution to this problem for the agricultural sector is the removal of infected tubers once the disease is detected, however, this does not guarantee that the disease hasn’t already spread to other plants. Research studies are underway to identify an effective control method for this pathogen, mainly by inov3PT (FN3PT research entity, the French agricultural professional organization for potato seeds.)

  It thus seemed highly relevant to us to carry out this proof of concept on Dickeya solani, to show that it is possible to follow the growth of the bacterium in situ on plants. This would enable researchers to efficiently study the effect of a possible treatment on potato plants, hence considerably accelerating the process and research currently being done.


  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).

Graphic of the optical density of the bacteria with the 4 different plasmids as a function of time

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

potato infections

Figure 5 - Infection of potato tubers by Dickeya solani containing pSEVA531 and pSEVA531-fiatlux


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

  • Citrobacter rodentium DBS100 (strain RLC2, NaIR) (see Material) is a laboratory mice pathogen. Bioluminescence imaging (BLI) has already been used to determine the in vivo colonization dynamics of C. rodentium. We decided to focus on this bacteria in order to enable future comparisons between our tool and other bioluminescent imaging solutions that exist. (Wiles and al. 2006)

  • Pseudomonas putida KT2440 (strain PP1) (see Material). It can be encountered in diverse ecological habitats. It has a remarkably versatile metabolism, adapted to withstand physicochemical stress, and the capacity to thrive in harsh environments. Owing to these characteristics, there is a growing interest in this microbe for industrial use, therefore, it seemed interesting for us to focus on this pathogen (Weimer et al. 2020).


Figure 6 - Results of the conjugation of pSEVA531 and pSEVA531-fiatlux in Citrobacter rodentium DBS100 (A & B) and in Pseudomonas putida KT2440 (C & D), on Petri dishes with 10µg/mL of tetracycline. Left (A & C): exposure time 0.1 sec; daylight. Right (B & D): exposure time 60 sec; without light. The disposition of plasmids is the same on all pictures.

  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)!


  Thanks to these results, we proved that our tool could be used with Dickeya solani, the bacteria responsible for causing blackleg disease on potatoes. Indeed, we proved that it did not affect their growth and made them luminescent. We also extended the proof of concept to two other bacterial strains, which also appeared luminescent.

  As a result, we proved that FIAT LUX is an efficient tool to track the path of bacteria in the plants and thus study infections, the first step towards finding solutions to fight these infections.

  This proof of concept shows that our project can honestly be a part of the solution to protect crops and secure a brighter future for global nutrition.


AHDB, Potato Disease Identification, 2022, available at:https://potatoes.ahdb.org.uk/knowledge-library/potato-disease-identification[Online Resource]

Blackleg of potato (Dickeya solani) | Department of Agriculture, Environment and Rural Affairs [WWW Document], 2017. DAERA. URL https://www.daera-ni.gov.uk/articles/blackleg-potato-dickeya-solani (accessed 9.7.22).

Dupuis, B., Nkuriyingoma, P., Van Gijsegem, F., 2021. Economic Impact of Pectobacterium and Dickeya Species on Potato Crops: A Review and Case Study. pp. 263–282. https://doi.org/10.1007/978-3-030-61459-1_8

Mansfield J, Genin S, Magori S, Citovsky V, Sriariyanum M, Ronald P, Dow M, Verdier V, Beer SV, Machado MA, Toth I, Salmond G, Foster GD. Top 10 plant pathogenic bacteria in molecular plant pathology. Mol Plant Pathol. 2012 Aug;13(6):614-29. doi: 10.1111/j.1364-3703.2012.00804.x. Epub 2012 Jun 5. PMID: 22672649; PMCID: PMC6638704.

Toth, I.K., van der Wolf, J.M., Saddler, G., Lojkowska, E., Hélias, V., Pirhonen, M., Tsror (Lahkim), L., Elphinstone, J.G., 2011. Dickeya species: an emerging problem for potato production in Europe. Plant Pathol. 60, 385–399. https://doi.org/10.1111/j.1365-3059.2011.02427.x

Weimer, Anna, Michael Kohlstedt, Daniel C. Volke, Pablo I. Nikel, et Christoph Wittmann. 2020. « Industrial Biotechnology of Pseudomonas Putida: Advances and Prospects ». Applied Microbiology and Biotechnology 104(18):7745 66. doi: 10.1007/s00253-020-10811-9

Wiles, Siouxsie, Karen M. Pickard, Katian Peng, Thomas T. MacDonald, et Gad Frankel. 2006. « In Vivo Bioluminescence Imaging of the Murine Pathogen Citrobacter Rodentium ». Infection and Immunity 74(9):5391 96. doi: 10.1128/IAI.00848-06.