The spontaneous self-assembly of individual components into complex structures underlies the synthesis of every system in nature at all scales. On a molecular scale, this process can assemble complete functional biomolecular machines out of hundreds of protein subunits, by coming together in just the right orientation at the right time without forming unwanted aggregates. Recent publications have indicated that this assembly process may be controlled by the use of a structural scaffold, and this mechanism has been proposed for a number of biomolecular machines, such as the Type III secretion system (T3SS), a bacterial syringe responsible for granting virulence to many gram-negative bacteria, including Shigella, Yersinia and Pseudomonas species. It is suspected that the needle of the T3SS acts as a structural scaffold for the tip complex, a pentameric protein structure responsible for initiation of host cell perforation and infection. We have endeavoured to replicate this assembly process with the use of a synthetic DNA scaffold, designed to match the structural properties of the natural scaffold, and present the first characterisation of DNA-templated cooperative synthesis of a protein complex using kinetics. We also present the first solution structure analysis of the T3SS tip proteins from five different bacterial species, which show an elongated domain not seen in the crystal structures that may be involved with the assembly process. These findings not only provide new insight into the mechanisms behind self-assembly of the T3SS tip complex, but large protein complexes in general, and paves a new way to synthesise biomolecular machines from the bottom up, for study and potential novel applications in bionanotechnology.