A Toolkit for Rapid Modular Construction of Biological Circuits in Mammalian Cells

João Pedro Fonseca; Alain R. Bonny; G. Renuka Kumar; Andrew H. Ng; Jason Town; Qiu Chang Wu; Elham Aslankoohi; Susan Y. Chen; Patrick Harrigan; Lindsey C. Osimiri; Amy L. Kistler; Hana El-Samad

Finally, while not explored in this work, the MTK constitutes a launching platform for additional exciting capabilities. For example, the MTK already expedites the production of sgRNA expression vectors, and with the prevalence of sgRNA screening approaches, we envision incorporating barcoding capabilities that can be read by bulk and single-cell sequencing technologies [36] [37] [38] . Furthermore, within the current MTK framework all TUs are read in the same direction. However, it is conceivable to design connector parts (1 and 5) that reverse the orientation of a TU to enable more autonomy in genetic circuit design and optimize transcriptional efficiency 33 . Lastly, the emergence of robotics and high-throughput liquid handling promises an exciting future avenue leveraging automation to streamline assembly. Overall, the MTK represents a significant advancement in cloning infrastructure to further expedite the growth, applications and scope of biological research and biotechnology. Thermocycler protocols: The "GG Long" protocol is primarily used for assembly reactions. The reaction temperature is initially held at 45°C for 2 min to digest the plasmids followed by 20°C . CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/506188 doi: bioRxiv preprint for 4 min to anneal constituent parts together. After repeating these first two steps 24 times, the temperature is increased to 60°C for 10 min to digest remaining recognition sites and inactivate the ligase. Then the temperature is held at 80°C for 10 min to inactivate the enzyme. Lastly, the reaction is held at 12°C indefinitely. The "GG End-On" protocol is used when BsaI or BsmBI sites need to be retained in the final product. The temperature is initially held at 45°C for 2 min to digest the plasmid followed by 20°C for 5 min to anneal and ligate the resulting plasmid.