TNT-Cloning: A New Platform for Flexible and Universal Usage of DNA Parts

Synthetic biology has developed rapidly due to our ability in performing deep-sequencing and synthesizing/assembling large fragments of DNA. However, the testing of several clusters for genetic circuits and pathway engineering is still costly and time consuming in the absence of a flexible, universal and all-inclusive methodology for joining DNA parts. Countless methods for in vivo and in vitro cloning were developed and different syntaxes were proposed, however, the natural course led the diverse synthetic biology community to adopt similar but yet distinct methodologies, suggesting the current available methods still lack coverage. Recently a modified typeIIS-based method called TNT-cloning was described showing unprecedented flexibility and universality for cloning DNA parts (De Paoli et al, 2016, Scientific Reports). Here we present a overview and comparison of the TNT-cloning versus other typeIIS-based methods and homology-based methods along with improvements performed in the published method as the inclusion of a lethal reporter (ccdB), for faster and more efficient cloning, as well as the putative expansion of the system to yeast and mammals aside E. coli and plants. The improved TNT-cloning system represents a step forward for supplying the synthetic biology community with a clean, efficient and malleable cloning system that has the potential to establish a quick and transferable platform necessary for determination of qualitative and quantitative gene fragment interactions that involves gene sets and gene networks in several fields.