A primary aim of Synthetic Biology is the design and implementation of biological systems that perform engineered functions. However, the assembly of double-stranded DNA molecules is a major barrier to this progress, as it remains time consuming and laborious. Here I present three improved methods for DNA assembly. The first is based on, and makes use of, BioBricks. The second method relies on overlap-extension PCR to assemble non-standard parts. The third method improves upon overlap extension PCR by reducing the number of steps and the time it takes to assemble DNA. Finally, I show how the PCR-based assembly methods presented here can be used, in concert, with in vivo homologous recombination in yeast to assemble as many as 19 individual DNA parts in one step. These methods will also be used to assemble an incoherent feedforward loop, gene regulatory network.

A primary aim of Synthetic Biology is the design and implementation of biological systems that perform engineered functions. However, the assembly of double-stranded DNA molecules is a major barrier to this progress, as it remains time consuming and laborious. Here I present three improved methods for DNA assembly. The first is based on, and makes use of, BioBricks. The second method relies on overlap-extension PCR to assemble non-standard parts. The third method improves upon overlap extension PCR by reducing the number of steps and the time it takes to assemble DNA. Finally, I show how the PCR-based assembly methods presented here can be used, in concert, with in vivo homologous recombination in yeast to assemble as many as 19 individual DNA parts in one step. These methods will also be used to assemble an incoherent feedforward loop, gene regulatory network.

The human central nervous system (CNS) contains a daunting number of cells and tremendous cellular diversity. A fundamental challenge of developmental neurobiology is to address the questions of how so many different types of neurons and glia can be generated at the precise time and place, making precisely the right connections. Resolving this issue involves dissecting the elaborate genetic networks that act within neurons and glia, as well as in the neural progenitor cells that generates them, to specify their identities. My PhD project has involved addressing a number of unresolved issues pertaining to how neural progenitor cells are specified to generate different types of neurons and glial cells in different temporal and spatial domains, and also how these early temporal and spatial cues are integrated to activate late cell fate determinants, which act in post-mitotic neural cells to activate distinct batteries of terminal differentiation genes. Analyzing the development of a specific Drosophila melanogaster (Drosophila) CNS stem cell – the neuroblast 5-6 (NB5-6) – we have identified several novel mechanisms of cell fate specification in the Drosophila CNS. We find that, within this lineage, the differential specification of a group of sequentially generated neurons – the Ap cluster neurons – is critically dependent upon the simultaneous triggering of two opposing feed-forward loops (FFLs) within the neuroblast. The first FFL involves cell fate determinants and progresses within the post-mitotic neurons to establish a highly specific combinatorial code of regulators, which activates a distinct battery of terminal differentiation genes. The second loop, which progresses in the neuroblast, involves temporal and sub-temporal genes that together oppose the progression of the first FFL. This leads to the establishment of an alternative code of regulators in late-born Ap cluster neurons, whereby alternative cell fates are specified. Furthermore, we find that the generation and specification of the Ap cluster neurons is modulated along the neuraxis by two different mechanisms. In abdominal segments, Hox genes of the Bithorax cluster integrates with Pbx/Meis factors to instruct NB5-6 to leave the cell cycle before the Ap cluster neurons are generated. In brain segments, Ap cluster neuron equivalents are generated, but improperly specified due to the absence of the proper Hox and temporal code. Additionally, in thoracic segments we find that the specification of the Ap cluster neurons is critically dependent upon the integration of the Hox, Pbx/Meis, and the temporal genes, in the activation of the critical cell fate determinant FFL. We speculate that the developmental principles of (i) feed-forward combinatorial coding; (ii) simultaneously triggered yet opposing feed-forward loops; and (iii) integration of different Hox, Pbx/Meis, and temporal factors, at different axial levels to control inter-segmental differences in lineage progression and specification; might be used widely throughout the animal kingdom to generate cell type diversity in the CNS.