Approaches for introducing large DNA molecules into bacterial cells

Engineering of the bacterial genome plays a key role in systems biology and synthetic biology. Genetic engineering of the bacterial genome involves the design and synthesis of large DNA molecules. However, functional studies of the designed and synthesized large DNA molecules are lagging. Methods for the transformation of large DNA molecules of bacterial chromosome size into bacterial cells through a single operation have not yet been established. Two major methods can be used for transferring large DNA molecules of bacterial chromosome size into bacterial cells: transformation mediated by liposomes or by microinjection. In both methods, cell wall (peptidoglycan layer)-deficient cells (l-form, protoplast, or spheroplast) should be used as the bacterial host cells. We succeeded in transferring a heterologous bacterial genome into an enlarged bacterial protoplast using a micromanipulator. This method for transferring large DNA molecules into bacterial cells through a single operation will contribute to both fundamental and applied research in microbial genome science.