Part:BBa_K2066002:Experience

Part of a suite of Iterative Capped Assembly (ICA) parts that can be used to assemble a TetO or LacO binding array of any size. These parts were designed based upon the ICA method of assembling repeat sequences from Briggs, et al. 2012 (“Iterative capped assembly: rapid and scalable synthesis of repeat-module DNA such as TAL effectors from individual monomers”). These assembled binding arrays can be used to horizontally shift the slope of a transfer function through molecular titration of transcription factors (LacI or TetR).

Additionally, there are 3 different verions of tetO ICA parts in this library, with either 8,16 or 64 base pair spacers between the tetO monomers, this is because it has been suggested by Amit et al. 2012 (“Building Enhancers from the Ground Up: A Synthetic Biology Approach” that anticooperativity plays a role in the effectivity of DNA protein binding. Anticoopritivity means that if two repressor binding sites are very close to each other, a repressor binding to one can spatially hinder, or even completely prevent a repressor from binding to the other. We thought that enabling another level of tuning to allow for even finer levels of shift magnitude by altering the level of anticooperativity would be a useful tool to include within the binding array section of the toolbox.

This part was sequence confirmed. Although a 3 TetO array was generated using this ICA library, we did not submit it because it would not have been capable of visibly shifting the transfer function of a genetic circuit.

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