De novo synthesis of a chloroplast genome in Nicotiana tabacum

Despite significant progress in chloroplast biotechnology the de novo synthesis of a chloroplast genome has

remained elusive. The development of strategies to design, assemble and transform artificial chloroplast genomes

would signify a step-change in the field of plant synthetic biology. Only recently, genome scale DNA synthesis

and sequencing has become routine, providing an ideal foundation to revisit the challenge of chloroplast genome

synthesis. I will join Ralph Bock, a world leader in chloroplast biotechnology, and combine the expertise of his

laboratory with my background in synthetic biology to generate the first Nicotiana tabacum plant with an artificial

chloroplast genome. I will base my strategy on creating and testing multiple different genome designs with varied

degrees of structural and genetic code alterations in respect to the parental genome to identify viable designs. I

will develop methods to assess those designs in parallel, permitting the rapid identification and fixing of

problematic regions in the synthetic genome. I will proceed to assemble the verified chloroplast genomes in a

hierarchical, parallelized manner from large pieces of synthetic DNA. Finally, I will characterize the phenotype of

the resulting plants, focusing on understanding the changes in transcription, translation and metabolism. The

methods developed in this work will provide insights into the design principles of organellar genomes, and provide

a platform to reinvent and remodel the genetic code of chloroplasts. This work will thereby establish strategies to

re-engineering complex cellular processes such as photosynthesis, or to design entirely new functions such as the

genetic isolation of synthetic DNA in plants.