Multi‐focused laboratory experiments based on Quorum Sensing and Quorum Quenching for acquiring Microbial Physiology concepts

After a time away from the classrooms and laboratories due to the global pandemic, the return to teaching activities during the semester represented a challenge to both teachers and students. Our particular situation in a Microbial Physiology course was the necessity of imparting in shorter time, laboratory practices that usually take longer. This article describes a 2‐week‐long laboratory exercise that covers several concepts in an interrelated way: conjugation as a gene transfer mechanism, regulation of microbial physiology, production of secondary metabolites, degradation of macromolecules, and biofilm formation. Utilizing a Quorum Quenching (QQ) strategy, the Quorum Sensing (QS) system of Pseudomonas aeruginosa is first attenuated. Then, phenotypes regulated by QS are evidenced. QS is a regulatory mechanism of microbial physiology that relies on signal molecules. QS is related in P. aeruginosa to several virulence factors, some of which are exploited in the laboratory practices presented in this work. QQ is a phenomenon by which QS is interrupted or attenuated. We utilized a QQ approach based on the enzymatic degradation of the P. aeruginosa QS signals to evidence QS‐regulated traits that are relevant to our Microbial Physiology course. Results obtained with the same test performed by a random group of students before and after the activities show the positive effectiveness of the approach presented in this work.

Other concepts (e.g., microbial growth, nutrition, and antibiosis) and skills (e.g., aseptic techniques, preparation of culture media, seeding, and incubation) are acquired in the General Microbiology course during the previous semester.
The global pandemic situation of COVID-19 that we have gone through from 2020 to 2021 has led us to rethink our traditional laboratory practices.In our faculty, the return to the in-person classes occurred in the middle of the semester.With fewer weeks for face-to-face teaching, teachers needed to achieve their study goals without lowering the quality of their teaching.At the same time, students had to acquire, in a short time, the corresponding knowledge related to this course.This challenge can be better faced if a common link that intersects the practical activities is found.The different aspects of microbial physiology are not independent but interconnected through regulatory mechanisms.This aspect is of particular relevance, considering that practical activities are sometimes imparted as independent units.As a consequence, we notice a failure to consider the cell as a whole, which is relevant in the biological sciences.Then, a concept unifying the independent practical activities is highly valuable.In certain microorganisms, Quorum Sensing (QS) systems are involved in the regulation of different physiological traits, many of which can be easily analyzed by undergraduate students.In this manner, several practical activities can be performed in a short time.Importantly, this approach teaches concretely that microbial physiology is a whole and not independent traits.
Employing Pseudomonas aeruginosa strain PAO1, we propose a series of lab practices in which students, in a short time, learn different concepts of microbial physiology in an interrelated manner: • Bacterial conjugation as a horizontal gene transfer mechanism.• Bioactive molecules.
• Signaling and regulation of microbial physiology through cell-to-cell communication.• Degradation of macromolecules.
Pseudomonas (P.) aeruginosa is a well-known Gram-negative pathogen widely spread in soil and aquatic environments. 1In P. aeruginosa PAO1, several virulence factors, including extracellular enzymes, production of biofilm, toxins, secondary metabolites, and siderophores, are regulated by its Quorum Sensing (QS) system. 2 QS is a cell density-dependent intercellular communication system that allows individual cells to act as a whole.QS mechanisms are based on the production, release, and detection of signal molecules called autoinducers. 3Similar to several other Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria, in P. aeruginosa these bioactive molecules are N-acyl homoserine lactones (AHLs). 2 Up to now, three QS systems intimately linked among them and arranged in a hierarchical regulatory cascade are known in P. aeruginosa.The autoinducer synthases LasI and RhlI catalyze the synthesis of the AHL molecules N-oxododecanoyl-L-homoserine lactone (3OC12-HSL) and N-butyryl-L-homoserine lactone (C4-HSL), respectively.When these autoinducers reach a critical threshold level by accumulation due to bacterial population growth, they interact with and are bound by their respective receptors, LasR and RhlR, which are at the same time transcriptional activators. 2 LasR and RhlR stimulate the lasI and rhlI expression, creating autoinduction loops.A third QS system that relies on the production of 2-heptyl-3-hydroxy-4-quinolone (Pseudomonas quinolone signal, or PQS) is interconnected with the LasI-LasR and RhlI-RhlR systems.
In nature, microorganisms interfere with the cellto-cell communication of competitors mainly through the enzymatic inactivation of the QS signals, or the production of metabolites that impede the normal functioning of the signal receptors, a phenomenon known as Quorum Quenching (QQ). 4 In the lab, QS systems can be blocked using QQ strategies to understand their functioning.In the approach chosen for the present work, the aiiA gene encoding the AiiA lactonase from Bacillus sp.A24, an AHL-degrading enzyme, is expressed under a constitutive P lac promoter in P. aeruginosa PAO1, reducing the accumulation of autoinducers and the expression of QSregulated traits. 5The easiest way to evidence the QQ phenomenon is to demonstrate the decrease in AHL levels employing biosensor strains.Biosensor strains have a genetic deficiency in signal production but detect and respond to exogenous autoinducers with a functional receptor, which is coupled to a reporter gene that produces an observable phenotype. 6,7Chromobacterium (C.) subtsugae CV026 and Pseudomonas putida F117 (pKR-C12) are the biosensors used in the practical activities presented in this work.Chromobacterium subtsugae CV026 produces violacein, a purple pigment, in the presence of AHLs with acyl side chains from C4 to C8 in length. 8P. putida F117 (pKR-C12) produces green fluorescent protein with AHLs with acyl side chains from C10 to C14 in length. 9aboratory activities are usually organized independently.In other words, though the different theoretical concepts are interconnected, the practical activities described in this work are usually imparted independently.The experimental workflow presented in this work is detailed in Figure 1.Each assay is thought of as a piece of a puzzle that fits together, giving a single, complete, overall, and multi-focused approach for the lab classes.For education purposes, P. aeruginosa PAO1 has the advantage that it is a fast-growing bacterium with well-known protocols for the evaluation of different physiological traits.However, it has to be highlighted that other microorganisms can be utilized if a characterized QS system is present.

| Education context
Our university is the National University of Tucuman (Universidad Nacional de Tucum an), located in the Argentine Northwest.Students belong not only to the province of Tucum an, but also to the neighboring provinces of Catamarca, Santiago del Estero, Salta and Jujuy.The Faculty of Biochemistry, Chemistry and Pharmacy (Facultad de Bioquímica, Química y Farmacia) also includes students for the Bachelor's degree in Biotechnology.Every year, around 45 students take the Microbial Physiology course.

| Equipment
The experimental procedures require the use of biosafety cabinets, micropipettes, centrifuges, vortex, UV transilluminator or a blue-light transilluminator, spectrophotometers, optical microscope, a rotary shaker, a rotary evaporator and a 37 C incubator.

| Biosafety
Even though these laboratory practices are not particularly risky, students need to be aware that they will be working with P. aeruginosa PAO1, a Biosafety Level 2 (BSL2) organism.Therefore, adequate laboratories with facilities for the development of the experiments presented in this work are required.In addition, all students should be instructed on proper laboratory safety practices and should follow established safety protocols.During all experiments, students must wear appropriate protective clothing, closed-toed shoes, and disposable gloves, and they should wash their hands at the end of each laboratory session.Surfaces or equipment must be wiped with disinfectant if contamination is suspected.Microbial cultures and reagents must be disposed of properly, and waste must be autoclaved before disposal.

| Strains and culture conditions
Bacterial strains used in this work are listed in Table 1.The culture media used for the assays are Luria Bertani (LB) broth composed of 10 g L À1 tryptone, 5 g L À1 yeast extract, 10 g L À1 NaCl; and nutrient yeast broth (NYB) composed of peptone 10 g L À1 ; NaCl 5 g L À1 ; meat extract 10 g L À1 ; yeast extract 5 g L À1 .The composition of Cetrimide agar utilized for selective isolation of transconjugants is gelatin peptone 20 g L À1 , MgCl 1.4 g L À1 , K 2 SO 4 10 g L À1 , centrimide 0.3 g L À1 , glycerol 10 mL L À1 .When required, culture media are solidified with agar 15 g L À1 and/or supplemented with tetracycline (Tc) 125 μg mL À1 sterilized by filtration through sterile 0.22 μm membrane filters.Pseudomonas aeruginosa PAO1 and the transconjugants are grown at 37 C and 180 rpm in an orbital shaker.Biosensor strains, C. subtsugae CV026 and P. putida F117 (pKR-C12) are grown in LB broth at 30 C and 180 rpm in an orbital shaker.
Pseudomonas aeruginosa PAO1 is available at the American Type Culture Collection (ATCC 15692).Chromobacterium subtsugae CV026 is available at the National Collection of Type Cultures (NCTC 13278).All other strains and plasmids are available from the authors of this work under the previous agreement of the original authors.

| Attenuation of Quorum Sensing activity
A QQ strategy is utilized to achieve the attenuation of P. aeruginosa PAO1 QS activity.Plasmid pME6863 contains Bacillus sp.A24 aiiA AHL-lactonase gene cloned in the pME6000 control vector. 5Both pME6863 and pME6000 are conjugated from Escherichia coli S17-1 into P. aeruginosa PAO1 by biparental mating, in two independent assays, as follows.Overnight cultures of P. aeruginosa PAO1, E. coli S17-1 (pME6000), and E. coli S17-1 (pME6863) on LB agar are utilized for the conjugation.For each conjugation, an inoculation loop of P. aeruginosa PAO1 is mixed with two loops of E. coli S17-1 (pME6000) or E. coli S17-1 (pME6863).Mixtures are prepared as small spots of 0.5-1 cm diameter on top of a LB agar plate.Conjugations are performed at 30 C for 24 h.Cell pellets are aseptically withdrawn, and suspended in saline.Serial dilutions prepared in this same solution are plated on Cetrimide agar plates supplemented with Tc for the selection of transconjugants.Cetrimide is a quaternary ammonium compound that inhibits E. coli strains; tetracycline allows the growth of only P. aeruginosa transconjugants.Isolated colonies are finally streaked on fresh cetrimide agar plates supplemented with Tc.

| AHLs production and extraction
Pseudomonas aeruginosa PAO1, P. aeruginosa PAO1 (pME6000), and P. aeruginosa PAO1 (pME6863) are independently pre-cultured for 18 h in NYB broth.These precultures are utilized to inoculate 125-mL flasks containing 20 mL of fresh culture media without antibiotics at an initial OD 600nm = 0.1.After 4 and 8 h, samples of 5 mL are withdrawn for AHLs extraction following the protocol described elsewhere. 10Briefly, one volume of ethyl acetate acidified with 0.1% acetic acid is mixed with one volume of cell-free supernatants.The samples are then vigorously extracted in a vortex for 5 min.After resting, the organic phase is removed, and a second extraction is performed from the aqueous phase.Both organic extractions are combined, and the solvent is evaporated to dryness in a rotary evaporator at 35-40 C. Finally, the extracts are solubilized in 10 μL acetonitrile.

| Bioassays
Ten milliliters of melted LB soft (0.6%) agar are independently seeded with overnight cultures of C. subtsugae CV026 and P. putida F117 (pKR-C12), and then poured on top of a layer of LB 1.5% agar.Two microliters of the organic extracts are spotted on top of the soft layer, and the solvent is left to evaporate.Plates are incubated at 30 C for 18-24 h.A positive result with C. subtsugae CV026 is evidenced by the presence of violet spots, which can be observed by the naked eye without the requirement of specific equipment.A positive result with P. putida F117 (pKR-C12) is evidenced by green fluorescent spots observed with a UV or a blue-light transilluminator.

| Evaluation of Quorum Sensingregulated phenotypes
In all assays, P. aeruginosa PAO1, P. aeruginosa (pME6000), and P. aeruginosa (pME6863) are grown T A B L E 1 Bacterial strains required for this laboratory work.

Chromobacterium subtsugae CV026
Biosensor of short-chain AHLs Pseudomonas putida F117 (pKR-C12) Biosensor of long-chain AHLs overnight in NYB broth, and then 5 μL are used to inoculate the corresponding plates.All incubations are carried out at 37 C for 24 h.Skim milk agar medium is used for the evaluation of protease production. 11A positive proteolytic activity is observed as clear halos due to the degradation of casein around the colonies.Interestingly, although King B medium is recommended for the evaluation of siderophore production, this phenotype can also be observed in skim milk agar (see below).Hemolytic activity is visualized on blood agar plates as clear halos around the colonies.In this medium, differences in the morphology of the colonies between the strains can also be observed.

| Implementation
The practice is proposed for 3 days over 2 weeks (Table 2).While the first week is planned for P. aeruginosa conjugations, the second week is used for the analysis of AHL production and the characterization of the phenotypes regulated by QS in the transconjugants.instance, the supplementation of the culture media with particular nutrients, the modification of incubation temperatures, or the presence of specific compounds in the assays can be easily implemented in the development of these experiments to evaluate the influence of different factors on QS-regulated traits.

| Assessment of laboratory activities
Students are assessed before the first and after the last laboratory activities.Before the activities, the instructor designs a short survey to assess the prior knowledge of the students about the topics to be covered during the laboratory activities.An example of a survey test is presented as Supplementary Material.In our University, this type of written test is usually evaluated by one instructor, which is responsible for a particular group of students.Students are expected to write and explain the key points in each question, not a complete description of each subject considered.A final laboratory report that includes all the observations, is prepared and evaluated after an oral presentation.A model of the Final Report is presented as Supplementary Material.According to our experience, we encourage the preparation of this report in groups of 2-3 students to promote the exchange of ideas and joint learning.This report allows the instructor to assess both the quality and interpretation of the results obtained and the ability of students to relate the experiments performed to the theoretical concepts.A model of grading rubric is also presented as Supplementary Material.

| RESULTS AND DISCUSSION
QS and QQ concepts are taught as part of General Microbiology, a previous course to Microbial Physiology.Experimental activities related to QS and QQ are usually not part of the Microbial Physiology course.However, its relevance in the regulation of different physiological traits of P. aeruginosa PAO1 can be exploited to teach different aspects of microbial physiology in an interconnected manner.

| First week
First day, Petri dishes containing sterile LB agar medium and overnight cultures of bacterial strains are available for conjugation assays (see Table 2).After a review of how to work employing aseptic techniques, students are organized in groups, as required.Conjugations with E. coli S17-1 strains carrying pME6000 or pME6863 are performed as described before.To note, tra transfer genes are integrated into the chromosome of E. coli S17-1, which allows biparental mating.Students also prepare cetrimide agar plates supplemented with Tc solution, which are required for the selection of transconjugants.The next day, students aseptically remove the bacterial growth from the plates where the conjugations were performed, and serial dilutions in saline are seeded on cetrimide agar plates with Tc.Plates are incubated at 37 C for 24 h.As negative controls, P. aeruginosa PAO1, E. coli S17-1 (pME6000), and E. coli S17-1 (pME6863) should also be plated on cetrimide agar with Tc.The next day, isolated colonies from biparental mating can be obtained.No colonies should be observed in the negative controls.At this point, a proper explanation of the importance of the composition of the culture medium and the complementation with Tc in the selection of transconjugants is critical.The instructor can also exemplify a hypothetical situation where no antibiotic or a non-selective medium (e.g., LB medium) is used in the selection of transconjugants.To note, cetrimide is the selective component that allows the growth of only P. aeruginosa, due to its surfactant activity.Finally, students select a plate with isolated colonies to continue the assays the following week.

| Second week
Previous to the lab session, liquid cultures of each strain are prepared as described above, and after 4 and 8 h of incubation, samples are withdrawn, which can be stored at À20 C until the organic extractions.First day, AHLs are extracted from supernatants with acidified ethyl acetate, and the extracts are concentrated to dryness.The instructor supports this experience by explaining the higher solubility of AHLs molecules in the organic phase, which allows their recovery during the extraction process.Students also prepare cultures of P. aeruginosa PAO1, P. aeruginosa PAO1 (pME6000), and P. aeruginosa PAO1 (pME6863) for assays of protease and hemolytic activities the next day, as well as cultures of C. subtsugae CV026 and P. putida F117 (pKR-C12) for bioassays.On the second day, students analyze the concentrated extracts using the two biosensor strains.Plates for protease and hemolytic activities are also seeded using overnight cultures of P. aeruginosa PAO1, P. aeruginosa PAO1 (pME6000) and P. aeruginosa PAO1 (pME6863).Ten microliters of each culture are seeded as equidistant spots on skim milk agar plates and blood agar plates.Finally, on the third day, students examine and discuss the results found in the bioassays (Figures 2 and 3) and the evaluation of the phenotypes (Figures 4 and 5).F I G U R E 5 Influence of QS on hemolytic activity on blood agar.Hemolysis is observed as clear halos around the colonies (A).Wider halos of hemolysis are observed with Pseudomonas aeruginosa PAO1 (a, A) and P. aeruginosa PAO1 (pME6000) (b, B), than with P. aeruginosa PAO1 (pME6863) (c, C).A direct observation of the colonies (right) allows also to distinguish differences in the morphologies of the colonies.
In the bioassays, the production of short-and longchain AHLs can be distinguished using C. subtsugae CV026 and P. putida F117 (pKR-C12) biosensor strains, respectively (see more details in Table 1).Students can also observe the effect of the QQ strategy on P. aeruginosa PAO1 (pME6863).The lactonase enzyme, coded in the pME6863 vector, degrades the synthesized AHLs; then, smaller or barely detectable spots in the bioassays are expected.It should be noted that the synthesis of AHLs is not blocked, but their accumulation is prevented by enzymatic degradation.The violet spots at the top of the plates in Figure 2A (samples obtained after 4 h of incubation), corresponding to P. aeruginosa PAO1 (left) and P. aeruginosa PAO1 (pME6000) (right), can be attributed to the induction of violacein production in C. subtsugae CV026 by C4-HSL.In the lower part of the plate, where the extract obtained from the P. aeruginosa PAO1 (pME6863) culture was seeded, the pigment production is not detected due to the degradation of the QS signal molecules.Figure 2B shows the results with samples obtained after 8 h of incubation.In this case, no differences are observed concerning the extracts obtained after 4 h of incubation, which contrasts when P. putida F117 (pKR-C12) biosensor strain is used.In this biosensor, 3OC12-HSL induces the production of green fluorescent protein (Figure 3).The analysis of the extracts of both P. aeruginosa PAO1 (left) and P. aeruginosa PAO1 (pME6000) (right) exhibits similar sizes of fluorescence spots.The small spot, corresponding to P. aeruginosa PAO1 (pME6863), is due to the lower levels of 3OC12-HSL related to the AHL degradation after 4 h of incubation (Figure 3A), and that is not detectable after 8 h of incubation (Figure 3B).The differences observed between the biosensor strains and the incubation time are related not only to the different levels of the AHLs but also to the different sensitivity to the QS signals.
Figure 4 shows the results obtained for protease activity.As expected from the bioassays, similar behaviors with P. aeruginosa PAO1 and P. aeruginosa PAO1 (pME6000) are observed.Both strains show positive protease activities evidenced by the presence of a clear halo around the colonies due to the casein degradation.Several genes coding for extracellular proteases are activated by QS in P. aeruginosa PAO1.LasA protease and LasB elastase are the best-characterized proteolytic enzymes and the ones that give the name to the LasI-LasR QS system. 12,13hough both Las and Rhl QS systems upregulate the expression of the corresponding lasA and lasB coding genes, the former is more prevalent than the latter.In addition, the green color of the colonies can be attributed to the siderophores production (Figure 4).Siderophores are organic iron chelators, which are also responsible for the typical green color to the Pseudomonas culture.
P. aeruginosa produces two siderophores in iron-limited media, named pyoverdine and pyochelin.pvd and pch genes, which are involved in pyoverdine and pyochelin synthesis, are upregulated by LasI-LasR. 14In P. aeruginosa PAO1 (pME6863), both protease activity and siderophore production are affected.
A similar situation is observed on blood agar plates, where positive hemolytic activities are detected for P. aeruginosa PAO1 and P. aeruginosa PAO1 (pME6000), but not for P. aeruginosa PAO1 (pME6863).Bacterial phospholipases are involved in hemolysis, though this activity is mainly related to the production of the surfactant rhamnolipids.Expression of rhlA and rhlB, which code for enzymes involved in rhamnolipid synthesis, is positively regulated by the RhlI-RhlR QS system. 15ifferences in the morphology of the colonies can also be observed on blood agar plates (Figure 5B-D).Bacterial colonies are static biofilms formed in the interphase between the culture medium and the air. 16A large set of genes is involved in biofilm formation in P. aeruginosa PAO1. 17Among them, alg genes involved in the production of the exopolysaccharide alginate, and the aforementioned rhlA and rhlB are upregulated by the RhlI-RhlR QS system. 15inally, the instructor may introduce students to the biotechnological applications derived from the use of QQ strategies for blocking cell-to-cell communication systems.Reference materials, including scientific articles, can support this discussion. 18,19Students are assessed through a final report covering the entire practical activities.

| Assessment of pedagogical effectiveness
To show the assessment of the experience presented in this work, we performed the same test before and after the practical activities.The evaluation scores (from 0 to 10) for each question of 11 randomly selected students are presented as a Supplementary table in Supplementary Material with mean values and standard deviations (see Supplementary Table S2).Same information is presented as a Supplementary figure (see Supplementary Figure S1).For the purpose of this work and, in order to avoid any bias in the scoring of the evaluations, these tests were evaluated by the same instructor.Scorings were finally checked by two authors of this work.Points were assigned considering the content and the use of technical vocabulary.A score of 10 was assigned when a complete and excellent response was presented; 0 was considered when a question was not responded.As shown, most of the questions were better responded to after the practical activities than before.The change in performance is relevant, considering the broad spectrum of concepts that are included in the practical activities.In addition, we noticed a notable positive attitude among the students not only during the practical activities but also during the discussions about the results obtained.These activities push the students to interconnect the different concepts, which is a practical and intellectual challenge.What is more, though the students do not perform scientific research during the development of these practical activities, the concept of "regulation" allows for discussion about other factors that could influence the Quorum Sensing activity, and other regulatory mechanisms that could be analyzed.

| Final considerations
Considering our curricula, experiments presented in this work allow us to integrate different concepts in a short period.Beyond the specific courses students must take at different universities and the specificities of each curriculum, activities presented in this work are common to other courses related to microbial physiology, as deduced from the contents of academic books utilized worldwide.In addition, relevant aspect of the experiments presented in this work is the potentiality of integrating different concepts using another central concept (in this case, QS and QQ) that encompasses them.Employing QS, other authors have proposed a series of experiments for students of different careers. 20In the present work, P. aeruginosa PAO1 is used as a model strain because its QS system is involved in the regulation of several readily demonstrable traits, including proteases and surfactants.These properties can be directly observed by students in simple and practical lab sessions.It has to be noted that the general concepts explored in these experiments are not specific to PAO1.Other bacteria with well-known QS systems can also be used, considering that the methodology should be adapted accordingly.However, in our opinion, P. aeruginosa PAO1 is an excellent choice due to the availability of protocols and the facility for the observation and interpretation of the results.These laboratory experiences allow, in a short time, to familiarize the students with several concepts and techniques, ranging from the regulation of genetic expression to the evidence of several physiological traits.

F
I G U R E 1 Workflow for the proposed laboratory class.Figure represents the individual tests to be performed to get a complete, overall, and multi-focused approach.

F I G U R E 2
Analysis of shortchain AHLs in extracts obtained from samples withdrawn after 4 h (A) and 8 h (B) of culture.Pseudomonas aeruginosa PAO1 (a), P. aeruginosa PAO1 (pME6000) (b), and P. aeruginosa PAO1 (pME6863) (c).Bioassay was developed using Chromobacterium subtsugae CV026.F I G U R E 3 Analysis of longchain AHLs in extracts obtained from samples withdrawn after 4 h (A) and 8 h (B) of culture.Pseudomonas aeruginosa PAO1 (a), P. aeruginosa PAO1 (pME6000) (b), and P. aeruginosa PAO1 (pME6863) (c).Bioassay was developed using Pseudomonas putida F177 (pKR-C12).Plates were observed in a blue light transilluminator.F I G U R E 4 Influence of QS on protease activity on skim milk agar.After 24 h of incubation, proteolytic activities are observed with Pseudomonas aeruginosa PAO1 (a) and P. aeruginosa PAO1 (pME6000) (b).Lower or no activity is observed with P. aeruginosa PAO1 (pME6863) (c).Production of siderophores (see the characteristic green color) is also observed in A and B.
Table2also shows the material required for each assay and the theoretical concepts to be learned that open the discussion and encourage critical thinking in the students.The class design is based on lab sessions lasting 4 h.At the beginning, an appropriate introductory explanation is provided by the instructor before starting the programmed activities.Then, students carry out the experimental part.Finally, the analysis and discussion of the results obtained are accomplished with the aim of helping students reinforce the concepts learned during the class.Potential pitfalls and solutions are presented in Supplementary TableS1.It has to be highlighted that, similar to other mechanisms that regulate microbial physiology, the activity of QS systems is influenced by both biotic and abiotic factors.This aspect can be exploited in the implementation of the experiments presented in this work for stimulate experimentation and scientific research in students.For T A B L E 2 Class design and organization of activities.