Self-regeneration is a fundamental function of all living systems. Here we demonstrate partial molecular self-regeneration in a synthetic cell. By implementing a minimal transcription- translation system within microfluidic reactors, the system is able to regenerate essential protein components from DNA templates and sustain synthesis activity for over a day. By quantitating genotype-phenotype relationships combined with computational modeling we find that minimizing resource competition and optimizing resource allocation are both criti- cally important for achieving robust system function. With this understanding, we achieve simultaneous regeneration of multiple proteins by determining the required DNA ratios necessary for sustained self-regeneration. This work introduces a conceptual and experi- mental framework for the development of a self-replicating synthetic cell.