A solid-phase platform for combinatorial and scarless multipart gene assembly

Abstract

With the emergence of standardized genetic modules as part of the synthetic biology toolbox, the need for universal and automatable assembly protocols increases. Although several methods and standards have been developed, these all suffer from drawbacks such as the introduction of scar sequences during ligation or the need for specific flanking sequences or a priori knowledge of the final sequence to be obtained. We have developed a method for scarless ligation of multipart gene segments in a truly sequence-independent fashion. The big advantage of this approach is that it is combinatorial, allowing the generation of all combinations of several variants of two or more modules to be ligated in less than a day. This method is based on the ligation of single-stranded or double-stranded oligodeoxynucleotides (ODN) and PCR products immobilized on a solid support. Different settings were tested to optimize the solid-support ligation. Finally, to show proof of concept for this novel multipart gene assembly platform a small library of all possible combinations of 4 BioBrick modules was generated and tested.