## 3 Biotechnological Approaches for Sensing and Actuation
### Biosensors for Environmental Monitoring
Cell group A provides sensing and a preliminary analysis of the measured signals and generates a chemical output that can be analyzed by the other groups of cells (cell groups B and C in Figure 3) for continuous processing and output generation. Next, some limitations and issues related to such sensor systems are discussed from the perspective of their possible application for a city's IoT:  
* There can be a long time between measuring substances arising, and response signals can result because of the need to provide many biosynthetic processes inside the cell, and this time can be equal to several hours [23];
* There is a possibility of situations that lead to massive cell deaths;
* There is intracellular resource competition--a lack of usable substrates that can prevent most of what is necessary for signal analysis biosynthesis and genetic logic circuit operation [73];
* There is less sensitivity in comparison with nanosized sensors or some electrochemical sensors;
* Difficulties occur with cell growth control and stabilization in an open environment, so the necessity of cell immobilization arises;
* Many cells influence the value of optical output, so an additional analysis of the measured data is needed to obtain reliable results;
* The long-time stable operation of such sensors in an open environment requires additional research;
* Legislation issues arise concerning the application of genetically modified organisms in an open environment, which also leads to the immobilization of cells and additional technical systems for preventing cell leakage [74]. Thus, some of those issues can be overcome using immobilization techniques [75] and using membranes between cells and the environment. Additionally, many studies have focused on the development of the modularity of engineered genetic circuits by adding retroactivity and feedback controllers to insulate modules [73; 76; 77]. The genetic logic circuit approach is not the only way to provide analysis using intracellular processes. Many studies have focused on analog synthetic biology [78], which has parallels between  
Figure 3: Multi-sensor cell systems with several stages of data processing. analog electric circuits and intracellular processing. This approach has many advantages such as the ability to develop hybrid systems with low-power electronics [79] and to develop new analog processes for measured data analysis; for example, some analogs of electrical circuit components have been developed [80]. It is also necessary to mention that the development of low-power cytomorphic electronics for biointerfaces is a promising tool for biosensors in IoT development [78; 79]. Next, attention will be paid to the approaches based on synthetic and modified proteins. The development of synthetic and modified proteins is an issue because there are multiple variants of ways in which protein can make its conformation, but this approach can yield new, more stable, and effective enzymes [81; 82] that can be used as the sensing element of biosensors. Protein systems can be used for intracellular computation [83]; for example, based on enzyme-catalyzed reactions, logic gates can be simulated [84]. A recent paper demonstrated the de novo design of protein logic gates that effectively operate in a leaving cell [85]. Protein logic gates are able to provide operations faster in comparison with genetic logic networks due to the possibility of the preliminary biosynthesis of several proteins that participate in the reactions. Finally, the interface between cells and our common electronic systems will be discussed. As shown in Figure 3, mainly fluorescent protein biosynthesis is used as an output signal [72]. This indicates the necessity of optical sensing systems. However, this does not mean that this is the only way this interface can be built. It is possible to use chemical signal-small chemical substances that can be easily detected and transferred to the electrical signal.