Synthetic biology is focused on the rational construction of biological systems based on engineering principles. During the field’s first decade of development, significant progress has been made in designing biological parts and assembling them into genetic circuits to achieve basic functionalities. These circuits have been used to construct proof-of-principle systems with promising results in industrial and medical applications. However, advances in synthetic biology have been limited by a lack of interoperable parts, techniques for dynamically probing biological systems, and frameworks for the reliable construction and operation of complex, higher-order networks. Here, we highlight challenges and goals for next-generation synthetic gene networks, in the context of potential applications in medicine, biotechnology, bioremediation, and bioenergy.