Abstract

The chloroplast is a highly attractive chassis for synthetic biology and metabolic engineering applications. To implement spatiotemporal control of gene expression in this organelle, we develop a synthetic transcriptional AND gate based on split T7 RNA polymerase (T7RNAP) in the simple model plant Marchantia polymorpha. A chloroplast-encoded fluorescent reporter under control of a T7 promoter will only be expressed if both the N- and C-terminal fragments of split T7RNAP , induced by two different input signals in the nucleus, are targeted to the chloroplast at the same time. As both the two independent input signals and the output of the AND gate may be chosen arbitrarily, this system may be adopted for various future applications in scope of developmental studies and metabolic engineering where spatiotemporal control of plastid gene expression is desired. Here, we report our progress in the design and characterization of the split T7RNAP system in Escherichia coli, and the identification of a functional chloroplast-targeting peptide in Marchantia polymorpha.