Breaking the limits of in vitro DNA Assembly
One research interest of the group is Golden Gate assembly, a DNA manipulation technique that allows users to efficiently stitch together multiple DNA fragments in a single tube. Golden Gate assembly uses a simple cut and paste workflow with a restriction enzyme to generate the DNA fragments, and a DNA ligase (usually T4 ligase) to join the fragments together. Our work in understanding ligase fidelity and bias has enabled us to design Golden Gate assemblies allowing unprecedented numbers of fragments (35+) to be assembled in a single reaction. We have used this approach to rapidly assemble 40-50kB bacteriophage genomes in vitro, and, in collaboration with the Ong and Tanner labs, now aim to show modular, hierarchical workflows permitting assembly of pooled oligos into 100kb+ constructs in 2-3 assembly rounds.
Current work seeks to apply new DNA assembly design rules and methods to increase the reliability of this work and allow even larger DNAs to be assembled along with the development of new protocols for efficient transformation of cells with these large synthetic constructs. Our ultimate aim is the parallelizable synthesis of full bacterial genomes, limiting in vivo steps prior to final transformation as much as possible.
Sikkema, A.P., et al (2023). Current Protocols. doi/10.1002/cpz1.882