###### Abstract  
Low-cost mini-bioreactor platforms are becoming increasingly important in synthetic biology and biotechnology for the characterization of genetic constructs and their initial scaling-up to large-scale cultures. Key process variables are the specific growth rate of cells and the synthesis rate of reporter proteins associated with transcriptional units of interest. These variables are of paramount importance for the characterization of gene synthetic circuits. In addition, their on-line estimation can be used for real-time monitoring of cells' metabolic state and gene circuit dynamic performance, thus allowing for on-line decision taking. In this work, we first describe a procedure for the calibration of absorbance and fluorescence measurements, ensuring standardized and comparable units across different experimental setups and measurement devices. Then, we implement an observer-based software sensor that uses the calibrated on-line measurements to simultaneously estimate the specific growth and protein synthesis rates. We implemented the calibration procedure and software sensor in a Chi.Bio mini-bioreactor platform. The experimental results show very good performance and pave the way for the use of mini-bioreactor platforms in the real-time characterization of gene synthetic circuits under dynamically regulated time-varying complex scenarios. s 2025 27 June 2 as industrial, experimental, and open-source platforms, are available on the market, each suited to different operational scales and research needs. While industrial bioreactors are typically designed for large-scale production processes, lab-scale systems are commonly used in laboratory environments to support research and development activities. Indeed, the possibility of setting culture conditions such as maintaining a steady cell growth rate or constant cell density, allows for the characterization of gene synthetic devices under a controlled physiological state of host-cells [10; 11]. Thus, constant cell density has allowed for the investigation of circuit performance under constant genetic load [12] and has been used to improve the predictability of gene synthetic devices [13]. On the other hand, the cell growth rate is directly linked to genetic noise [14] and affects the dynamics of gene expression. Indeed, the cell growth rate is representative of the cell metabolic state [15]. The growth rate reflects how cells allocate their metabolic resources and thus directly affects the behavior of synthetic circuits. Thus, control of the cell growth rate ensures that observed behaviors are not consequences of uncontrolled metabolic fluctuations and allows for the precise characterization and more accurate parameter fitting in mathematical models of gene regulation [16]. In this context, small-scale open-source bioreactors are increasingly being used for the characterization of gene synthesis devices under different culture environments, and for their design in the framework of Design-Build-Test-Learn (DBTL) iterative strategies [17; 18; 19; 20]. Mini-bioreactors are small-volume, highly automated cultivation systems (typically 5-50 mL) that replicate the control features of large-scale fermenters. Their use of mini-bioreactors offers important advantages for the reliable characterization of genetic circuits over lab-scale and larger ones [21]. Mini-bioreactors enable parallel experiment to be used under precisely controlled conditions, which is ideal for characterizing libraries of genetic devices under uniform metabolic states. They can mimic chemostat or turbidostat conditions at a small scale, with reduced reagent cost and volume, and yet be informative towards scaling-up [22]. Most modern bioreactors have sensors that monitor key variables of bacterial cultures, such as temperature, pH, absorbance, and fluorescence.