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The reconstruction of biological processes in vitro offers a powerful route towards the bottom-up construction of synthetic cells. However, the complex dynamics of biological systems make their emulation in synthetic systems a considerable challenge. Here, we present a partially self-regenerating minimal cell consisting of a microfluidic reactor hosting a cell-free gene expression system, which is periodically replenished with small volumes of feeding solution. This system sustains the expression of several essential protein components, including ribosomal proteins, translation factors, and T7 RNA polymerase, over a 24-h period. By maintaining a balance between supply and removal, and by avoiding energy depletion and component degradation, the system exhibits an increase in self-regeneration capacity. Our work demonstrates a step towards the sustained operation of synthetic minimal cells.  +