# A Microbial Screening in Silico Method for the Fitness Step Evaluation in Evolutionary Algorithms
## 2 Materials and Methods
### Microbial Screening Methods in Silico: Procedures and Implementation  
Microbial screening procedures in silico are algorithms simulating biological mechanisms and experimental protocols that are usually applied in the wet lab. In order for a screening routine to be capable of detecting and evaluating the fitness of a given candidate solution, we will need a feature which value increases according to the suitability of the solution. In our case, the size of the colony (i.e., the number of synthetic bacteria), the total expression of a reporter protein, e.g., green fluorescent protein (GFP), etc. In this work, we designed three screening procedures (Figure 3) based on the following bacterial features: (a) bacterial resistance to an antibiotic, (b) processing of lactose (i.e., bacteria use this sugar as an energy source) by Lac operon and (c) chemotaxis, i.e., the ability of bacteria to move to a food source or other attractive substance. In the following, we describe the basis of the three screening routines, whereas the details of the scripts, i.e., their implementation in the Gro language, are described in the Appendix A:  
* Antibiotic resistance: This selection method is the usual one in a laboratory of microbiology, synthetic biology, etc., since it is easy to apply and resistance is known for a multitude of antibiotics [20]. The implementation in Gro (Appendix A, Figure A1) of this screening procedure simulates a randomly distributed signal emulating an antibiotic (Figure 4), being received by all synthetic bacteria. Since in Gro there is a command to cause the death of bacterial cells,  
Figure 3: Screening procedures developed in Gro language. (**a**) Antibiotic resistance, (**left**) detail of an antibiogram and (**right**) its simulation. (**b**) Lac operon, (**left**) A MacConkey agar plate with a bacterial culture of microbes fermenting lactose and (**right**) its simulation. (**c**) Chemotaxis, (**left**) _Pseudomonas aeruginosa_ swarming motility and (**right**) its simulation. this command is used to simulate the effect of the antibiotic on those bacteria that do not satisfy a particular logical condition. Thus, all synthetic bacteria with the desired phenotype will be resistant to the signal, while the rest of the bacteria will die. * Lac Operon: The genes of bacteria are organized into operons [21]. When nutrient levels in the environment change, the genes of the bacteria vary in their expression in response to those changes. For example, the operon lactose (Lac) changes its state depending on the levels of lactose sugar in the environment. In the experimental realm, the Lac operon is one of the most commonly used in synthetic biology experiments, contributing to the controlled and effective implementation of diverse biotechnological processes. At the cellular level, the Lac operon is in charge of regulating the synthesis of the molecular machinery necessary for lactose degradation. Therefore, its presence in the bacteria is an advantage that can be used in a selection procedure. The Lac operon implementation in Gro (Appendix A, Figure A2) is inspired by the logic that regulates the operon (Figure 5), not including the regulating elements of the operon. In this work, we have only simulated the conditions under lacZ effector acts on the operon: in the absence or low levels of glucose and presence of lactose. In the Gro routine, such conditions will be subrogated to the logical condition of the optimization problem to be solved. Therefore, if the synthetic bacterium does not meet the logical condition of the problem, the bacterium will not hold the operon, and therefore will not be able to degrade the lactose. In addition, as the bacterial growth rate is related to the amount of available nutrients, bacterial colonies unable to degrade lactose will have a lower fitness value. In this screening procedure, besides the logic described above, the different metabolic reactions that occur during the degradation of the main carbon source were also taken into account.