Advancements in synthetic biology-based bacterial cancer therapy: A modular design approach.

Synthetic biology aims to program living bacteria cells with artificial genetic circuits for user-defined functions, transforming them into powerful tools with numerous applications in various fields, including oncology. Cancer treatments have serious side effects on patients due to the systemic action of the drugs involved. To address this, new systems that provide localized antitumoral action while minimizing damage to healthy tissues are required. Bacteria, often considered pathogenic agents, have been used as cancer treatments since the early 20th century. Advances in genetic engineering, synthetic biology, microbiology, and oncology have improved bacterial therapies, making them safer and more effective. Here we propose six modules for a successful synthetic biology-based bacterial cancer therapy, the modules include Payload, Release, Tumor-targeting, Biocontainment, Memory, and Genetic Circuit Stability Module. These will ensure antitumor activity, safety for the environment and patient, prevent bacterial colonization, maintain cell stability, and prevent loss or defunctionalization of the genetic circuit.

PLATERO: A calibration protocol for plate reader green fluorescence measurements.

One of the most common sources of information in Synthetic Biology is the data coming from plate reader fluorescence measurements. These experiments provide a measure of the light emitted by a certain fluorescent molecule, such as the Green Fluorescent Protein (GFP). However, these measurements are generally expressed in arbitrary units and are affected by the measurement device gain. This limits the range of measurements in a single experiment and hampers the comparison of results among experiments. In this work, we describe PLATERO, a calibration protocol to express fluorescence measures in concentration units of a reference fluorophore. The protocol removes the gain effect of the measurement device on the acquired data. In addition, the fluorescence intensity values are transformed into units of concentration using a Fluorescein calibration model. Both steps are expressed in a single mathematical expression that returns normalized, gain-independent, and comparable data, even if the acquisition was done at different device gain levels. Most important, the PLATERO embeds a Linearity and Bias Analysis that provides an assessment of the uncertainty of the model estimations, and a Reproducibility and Repeatability analysis that evaluates the sources of variability originating from the measurements and the equipment. All the functions used to build the model, exploit it with new data, and perform the uncertainty and variability assessment are available in an open access repository.

Multicolor plate reader fluorescence calibration.

Plate readers are commonly used to measure cell growth and fluorescence, yet the utility and reproducibility of plate reader data is limited by the fact that it is typically reported in arbitrary or relative units. We have previously established a robust serial dilution protocol for calibration of plate reader measurements of absorbance to estimated bacterial cell count and for green fluorescence from proteins expressed in bacterial cells to molecules of equivalent fluorescein. We now extend these protocols to calibration of red fluorescence to the sulforhodamine-101 fluorescent dye and blue fluorescence to Cascade Blue. Evaluating calibration efficacy via an interlaboratory study, we find that these calibrants do indeed provide comparable precision to the prior calibrants and that they enable effective cross-laboratory comparison of measurements of red and blue fluorescence from proteins expressed in bacterial cells.

Modeling and Optimization of a Molecular Biocontroller for the Regulation of Complex Metabolic Pathways.

Achieving optimal production in microbial cell factories, robustness against changing intracellular and environmental perturbations requires the dynamic feedback regulation of the pathway of interest. Here, we consider a merging metabolic pathway motif, which appears in a wide range of metabolic engineering applications, including the production of phenylpropanoids among others. We present an approach to use a realistic model that accounts for in vivo implementation and then propose a methodology based on multiobjective optimization for the optimal tuning of the gene circuit parts composing the biomolecular controller and biosensor devices for a dynamic regulation strategy. We show how this approach can deal with the trade-offs between the performance of the regulated pathway, robustness to perturbations, and stability of the feedback loop. Using realistic models, our results suggest that the strategies for fine-tuning the trade-offs among performance, robustness, and stability in dynamic pathway regulation are complex. It is not always possible to infer them by simple inspection. This renders the use of the multiobjective optimization methodology valuable and necessary.

Multi-Objective Optimization Tuning Framework for Kinetic Parameter Selection and Estimation.

Semi-mechanistic kinetic (i.e., dynamic) models based on first principles are particularly relevant in biology, as they can explain and predict functional behavior that arises from varying concentrations of the cellular components over time. Here, we describe a computational tuning framework to facilitate both the selection of kinetic parameters for these models and its estimation from experimental data. On the one hand, the tuning framework uses multi-objective optimization to generate a model-based set of guidelines for the selection of the kinetic parameters. These parameter values are the required ones to provide a biological system with desired behavior, while fulfilling the design criteria encoded in the optimization problem itself. On the other hand, this framework can also be used to estimate the parameter values of biological systems from experimental data, once the optimization objectives had been defined appropriately. The methodology gives accurate identification results, as it provides clear orientation on the effect of the parameter values over the system's behavior even under different experimental scenarios. It is particularly useful for easily combining time-course-averaged data and steady-state distribution data. This protocol also addresses aspects related to the appropriate description of the kinetic models and the settings of the software tools. Therefore, it supplies for hands-on testing to evaluate the validity of the underlying technical assumptions of the biological kinetic models.

RBS and Promoter Strengths Determine the Cell-Growth-Dependent Protein Mass Fractions and Their Optimal Synthesis Rates.

Models of gene expression considering host-circuit interactions are relevant for understanding both the strategies and associated trade-offs that cell endogenous genes have evolved and for the efficient design of heterologous protein expression systems and synthetic genetic circuits. Here, we consider a small-size model of gene expression dynamics in bacterial cells accounting for host-circuit interactions due to limited cellular resources. We define the cellular resources recruitment strength as a key functional coefficient that explains the distribution of resources among the host and the genes of interest and the relationship between the usage of resources and cell growth. This functional coefficient explicitly takes into account lab-accessible gene expression characteristics, such as promoter and ribosome binding site (RBS) strengths, capturing their interplay with the growth-dependent flux of available free cell resources. Despite its simplicity, the model captures the differential role of promoter and RBS strengths in the distribution of protein mass fractions as a function of growth rate and the optimal protein synthesis rate with remarkable fit to the experimental data from the literature for Escherichia coli. This allows us to explain why endogenous genes have evolved different strategies in the expression space and also makes the model suitable for model-based design of exogenous synthetic gene expression systems with desired characteristics.

Stochastic Differential Equations for Practical Simulation of Gene Circuits.

The Chemical Langevin Equation approach allows simple stochastic simulation of gene circuits under many practical situations where the number of molecules of the species involved is not extremely low. Here, we describe methods and a computational framework to simulate a population of cells containing gene circuits of interest. These methods account for both intrinsic and extrinsic noise sources, and allow us to have both individual cell-related species and population-related ones. The protocol covers aspects related to proper description of the system and setting the software tools. It also helps to deal with the optimization of data storage and the simulation precision versus computational time issue. Finally, it also gives practical tests to assess the validity of the underlying technical assumptions.

Extended Metabolic Biosensor Design for Dynamic Pathway Regulation of Cell Factories.

Transcription factor-based biosensors naturally occur in metabolic pathways to maintain cell growth and to provide a robust response to environmental fluctuations. Extended metabolic biosensors, i.e., the cascading of a bio-conversion pathway and a transcription factor (TF) responsive to the downstream effector metabolite, provide sensing capabilities beyond natural effectors for implementing context-aware synthetic genetic circuits and bio-observers. However, the engineering of such multi-step circuits is challenged by stability and robustness issues. In order to streamline the design of TF-based biosensors in metabolic pathways, here we investigate the response of a genetic circuit combining a TF-based extended metabolic biosensor with an antithetic integral circuit, a feedback controller that achieves robustness against environmental fluctuations. The dynamic response of an extended biosensor-based regulated flavonoid pathway is analyzed in order to address the issues of biosensor tuning of the regulated pathway under industrial biomanufacturing operating constraints.

Characterization of Gene Circuit Parts Based on Multiobjective Optimization by Using Standard Calibrated Measurements.

Standardization and characterization of biological parts is necessary for the further development of bottom-up synthetic biology. Herein, an easy-to-use methodology that embodies both a calibration procedure and a multiobjective optimization approach is proposed to characterize biological parts. The calibration procedure generates values for specific fluorescence per cell expressed as standard units of molecules of equivalent fluorescein per particle. The use of absolute standard units enhances the characterization of model parameters for biological parts by bringing measurements and estimations results from different sources into a common domain, so they can be integrated and compared faithfully. The multiobjective optimization procedure exploits these concepts by estimating the values of the model parameters, which represent biological parts of interest, while considering a varied range of experimental and circuit contexts. Thus, multiobjective optimization provides a robust characterization of them. The proposed calibration and characterization methodology can be used as a guide for good practices in dry and wet laboratories; thus allowing not only portability between models, but is also useful for generating libraries of tested and well-characterized biological parts.

Engineered Control of Genetic Variability Reveals Interplay among Quorum Sensing, Feedback Regulation, and Biochemical Noise.

Stochastic fluctuations in gene expression trigger both beneficial and harmful consequences for cell behavior. Therefore, achieving a desired mean protein expression level while minimizing noise is of interest in many applications, including robust protein production systems in industrial biotechnology. Here, we consider a synthetic gene circuit combining intracellular negative feedback and cell-to-cell communication based on quorum sensing. Accounting for both intrinsic and extrinsic noise, stochastic simulations allow us to analyze the capability of the circuit to reduce noise strength as a function of its parameters. We obtain mean expression levels and noise strengths for all species under different scenarios, showing good agreement with system-wide available experimental data of protein abundance and noise in Escherichia coli. Our in silico experiments, validated by preliminary in vivo results, reveal significant noise attenuation in gene expression through the interplay between quorum sensing and negative feedback and highlight the differential role that they play in regard to intrinsic and extrinsic noise.

Multi-objective optimization framework to obtain model-based guidelines for tuning biological synthetic devices: an adaptive network case.

BACKGROUND: Model based design plays a fundamental role in synthetic biology. Exploiting modularity, i.e. using biological parts and interconnecting them to build new and more complex biological circuits is one of the key issues. In this context, mathematical models have been used to generate predictions of the behavior of the designed device. Designers not only want the ability to predict the circuit behavior once all its components have been determined, but also to help on the design and selection of its biological parts, i.e. to provide guidelines for the experimental implementation. This is tantamount to obtaining proper values of the model parameters, for the circuit behavior results from the interplay between model structure and parameters tuning. However, determining crisp values for parameters of the involved parts is not a realistic approach. Uncertainty is ubiquitous to biology, and the characterization of biological parts is not exempt from it. Moreover, the desired dynamical behavior for the designed circuit usually results from a trade-off among several goals to be optimized. RESULTS: We propose the use of a multi-objective optimization tuning framework to get a model-based set of guidelines for the selection of the kinetic parameters required to build a biological device with desired behavior. The design criteria are encoded in the formulation of the objectives and optimization problem itself. As a result, on the one hand the designer obtains qualitative regions/intervals of values of the circuit parameters giving rise to the predefined circuit behavior; on the other hand, he obtains useful information for its guidance in the implementation process. These parameters are chosen so that they can effectively be tuned at the wet-lab, i.e. they are effective biological tuning knobs. To show the proposed approach, the methodology is applied to the design of a well known biological circuit: a genetic incoherent feed-forward circuit showing adaptive behavior. CONCLUSION: The proposed multi-objective optimization design framework is able to provide effective guidelines to tune biological parameters so as to achieve a desired circuit behavior. Moreover, it is easy to analyze the impact of the context on the synthetic device to be designed. That is, one can analyze how the presence of a downstream load influences the performance of the designed circuit, and take it into account.