Bacterial microcompartments are proteinaceous complexes that catalyze metabolic pathways in a manner reminiscent of organelles. Although microcompartment structure is well understood, much less is known about their assembly and function in vivo. We show here that carboxysomes, CO 2 -fixing microcompartments encoded by 10 genes, can be heterologously produced in Escherichia coli. Expression of carboxysomes in E. coli resulted in the production of icosahedral complexes similar to those from the native host. In vivo, the complexes were capable of both assembling with carboxysomal proteins and fixing CO 2 . Characterization of purified synthetic carboxysomes indicated that they were well formed in structure, contained the expected molecular components, and were capable of fixing CO 2 in vitro. In addition, we verify association of the postulated pore-forming protein CsoS1D with the carboxysome and show how it may modulate function. We have developed a genetic system capable of producing modular carbon-fixing microcompartments in a heterologous host. In doing so, we lay the groundwork for understanding these elaborate protein complexes and for the synthetic biological engineering of self-assembling molecular structures.ribulose 1,5-bisphosphate carboxylase/oxygenase | synthetic biology | metabolic engineering | self-assembly

While complex dynamic biological networks control gene expression in all living organisms, the forward engineering of comparable synthetic networks remains challenging. The current paradigm of characterizing synthetic networks in cells results in lengthy design-build-test cycles, minimal data collection, and poor quantitative characterization. Cell-free systems are appealing alternative environments, but it remains questionable whether biological networks behave similarly in cell-free systems and in cells. We characterized in a cell-free system the ‘repressilator’, a three-node synthetic oscillator. We then engineered novel three, four, and five-gene ring architectures, from characterization of circuit components to rapid analysis of complete networks. When implemented in cells, our novel 3-node networks produced population-wide oscillations and 95% of 5-node oscillator cells oscillated for up to 72 hr. Oscillation periods in cells matched the cell-free system results for all networks tested. An alternate forward engineering paradigm using cell-free systems can thus accurately capture cellular behavior.DOI: http://dx.doi.org/10.7554/eLife.09771.001

Living cells maintain a steady state of biochemical reaction rates by exchanging energy and matter with the environment. These exchanges usually do not occur in in vitro systems, which consequently go to chemical equilibrium. This in turn has severely constrained the complexity of biological networks that can be implemented in vitro. We developed nanoliter-scale microfluidic reactors that exchange reagents at dilution rates matching those of dividing bacteria. In these reactors we achieved transcription and translation at steady state for 30 h and implemented diverse regulatory mechanisms on the transcriptional, translational, and posttranslational levels, including RNA polymerases, transcriptional repression, translational activation, and proteolysis. We constructed and implemented an in vitro genetic oscillator and mapped its phase diagram showing that steady-state conditions were necessary to produce oscillations. This reactor-based approach will allow testing of whether fundamental limits exist to in vitro network complexity.synthetic biology | cell-free protein synthesis | computational biology | minimal artificial cell I nstead of complex and ill-characterized cellular hosts, in vitro systems have recently become popular alternatives for implementing synthetic networks. In vitro systems can be completely defined, easily manipulated, interrogated, and have been used to study a number of biological phenomena. For example, periodic temporal patterns were observed in systems based on nucleic acid synthesis and degradation (1, 2), and ordered spatial patterns were created from purified cell division regulators (3). In vitro transcription and translation (ITT)-based systems should, in principle, be able to use all regulatory functionalities found in living cells. Reconstituted, defined ITT systems like the PURE mix (4), are particularly appealing for bottom-up synthetic biology. A number of recent examples show that various genetic (5-10) and metabolic (11) networks can be implemented in ITT systems. Genetic network complexity has, however, been limited to genetic cascades, where the output of one stage acts on the next stage, whereas examples of positive and negative feedback have been basic (8,9,12). The main limitation to network complexity in vitro derives from its batch reaction format. In batch, synthesis rates decrease as precursors are consumed, enzymatic activities degrade, and reaction products accumulate. This rapid approach to chemical equilibrium severely limits network size. In addition, negative feedback is particularly difficult to implement, because regulators from earlier stages are not removed. The implementation of active degradation mechanisms for RNA and proteins (13) could solve the problem of product removal, and synthesis times can be increased by using reactors that allow an exchange of small molecules between the ITT mix and a feeding solution. Large-volume continuous flow and exchange systems were developed to increase the amounts of protein produced by ITT systems and are based on di...