Initial steps towards the establishment of a Multigene Engineering Platform in

Drosophila melanogaster

Alejandro Sarrion-Perdigones1, Nicholas Abel2, Nick Matinyan3, Kristi L. Hoffman1, Audrey E. Christiansen1, Koen J.T. Venken1,2,3,4,5.

1 Verna and Marrs McLean Department of Biochemistry and Molecular Biology

2 Department of Pharmacology

3 Program in Integrative Molecular and Biomedical Sciences

4 Dan L. Duncan Cancer Center

5 Program in Developmental Biology

Baylor College of Medicine, TX, 77030

The application of synthetic biology principles for the analysis of biology and disease in multicellular organisms is a recently growing field. Currently there is no standard DNA assembly methodology to facilitate multigene engineering in Drosophila melanogaster. To overcome the existing limitations on the design and construction of new genetic combinations in this model organism and enable the exchange of genetic building blocks, we propose the establishment of a platform founded on the synergistic combination of two transgenesis technologies: GoldenBraid2.0, a synthetic assembly platform that facilitates multigene engineering via Type IIs restriction enzyme assembly, and P[acman], the high-capacity site-specific transgenesis platform developed for Drosophila melanogaster. Apart from providing an optimized cloning strategy that generates fully exchangeable genetic elements for multigene engineering, the platform will include the most advanced tools for genetic engineering in Drosophila. Our broad technological framework aims to establish the foundations of Synthetic Biology in the fly field and can be easily extrapolated towards other model organisms.
Here we present an initial group of elements characterized in Drosophila Schneider 2 (S2) cells. These elements include a strategically chosen group of functionally-tested, pre-made genetic parts to build frequently-used modules such as constitutive, tissue-specific and inducible expression cassettes as well as fluorescent protein and other reporter units common to Drosophila research. These elements are components of an ever-growing open collection of DNA parts that will be available to any interested researcher. This platform, in combination with other shared resources in the fly community (e.g., Bloomington Drosophila Stock Center, BACPAC Resources, Drosophila Genomics Resource Center, and Addgene), will facilitate the expansion of complex synthetic biology advances in Drosophila melanogaster.