# Evolutionary Algorithms in a Bacterial Consortium of Synthetic Bacteria
## 2 Materials and Methods
### A Preliminary Experiment for Bottom-Up Design of Synthetic Bacteria: GADY  
The present work originates from a simulation experiment carried out some time ago and recently published as a pre-print [20]. In order to carry out the study in silico of a microbial community or consortium as simply as possible, we performed a preliminary experiment that we termed GADY (an acronym for get signal; antidote; die when a killer gene is expressed; yellow fluorescence). Since the microbial community was the simplest possible, we assumed a microbial consortium consisting of only two bacterial species: one pathogenic and the other non-pathogenic. The pathogenicity or not of the bacteria was only a label since the aim of this study was not the simulation of a disease caused by bacterial infection. The goal of the experiment was to evaluate an evolutionary algorithm oriented to the eradication of the pathogenic bacterium present in a microbial consortium. In this model, only pathogenic bacteria undergo Darwinian natural selection, while the non-pathogenic strain remains in the consortium without evolutionary changes. The algorithm makes the assumption that bacteria communicate with each other by quorum sensing, i.e., a type of communication between bacteria that takes place, for example, in the bacterial strain _E. coli_. At the beginning of the simulation experiment (\(t=0\)), there is a single initial cell or bacterium that divides into two daughter bacteria (Figure 2C). One of the daughter bacteria behaves as non-pathogenic, which sends a chemical signal, while the other daughter bacterium is the pathogenic bacterium that receives the signal (Figure 3). In order to introduce genetic variability in the microbial community, the experiment was conducted by programming synthetic pathogenic bacteria with the genetic mechanism of mutation. As mentioned above, communication between non-pathogenic and pathogenic bacteria occurs in the microbial community through a signaling mechanism known as quorum sensing. This form of communication, which uses AHL (N-acyl homoserine lactone) as a signaling molecule, is simulated as follows. The genome of the sender bacteria (non-pathogenic) includes a plasmid that is composed of a single luxl gene (Figure 3), whereas the receptor bacteria (pathogenic) host a plasmid consisting of four genes: luxR, killer antidote, killer, and yfp (Figure 3). Luxl in the sender bacterium is responsible for producing AHL, releasing this molecule to the environment. AHL enters into the pathogenic bacterium and binds to luxR, forming a complex. This is followed by the expression of the remaining genes except for the killer gene, whose expression is inhibited by the killer antidote. In the case of the yfp gene, its expression results in the synthesis of the yellow fluorescent protein, resulting in the emission of yellow fluorescence from the bacteria. The pathogenic bacterium has the luxR gene capable of detecting the AHL inducer signal, i.e., the signal that has been emitted by the non-pathogenic bacterium. Figure 3: GADY genetic network. (**Left**) AHL-emitting bacterium (‘green’ bacterium is the non-pathogenic one) with a plasmid including Luxl gene. (**Right**) Representation of the gene circuit of the plasmid hosted in the ‘red’ pathogenic bacterium. Figure 2: Models of consortia or microbial communities composed of four strains or bacterial species (**B1–B4**). Bacterial reproduction occurs in each colony by bipartition; however, some colonies yield pathogenic daughter bacteria, others yield non-pathogenic daughter bacteria, and there are colonies in which there are bacteria of both cell lines. Bacteria displayed in ‘green’ are non-pathogenic, while ‘red’ bacteria are pathogenic. (**A**) Experiments 1 and 2. (**B**) Experiment 3. (**C**) GADY experiment. In this case, the simulation experiment was conducted [20] with a single colony of bacteria of the same strain and not with a bacterial community. (**D**) Experiment 4 (for a detailed explanation of the simulation experiments, see text).