Recursive Directional Ligation by Plasmid Reconstruction Allows Rapid and Seamless Cloning of Oligomeric Genes

Abstract

This paper reports a new strategy, recursive directional ligation by plasmid reconstruction (PRe-RDL), to rapidly clone highly repetitive polypeptides of any sequence and specified length over a large range of molecular weights. In a single cycle of PRe-RDL, two halves of a parent plasmid, each containing a copy of an oligomer, are ligated together, thereby dimerizing the oligomer and reconstituting a functional plasmid. This process is carried out recursively to assemble an oligomeric gene with the desired number of repeats. PRe-RDL has several unique features that stem from the use of type IIs restriction endonucleases: first, PRe-RDL is a seamless cloning method that leaves no extraneous nucleotides at the ligation junction. Because it uses type IIs endonucleases to ligate the two halves of the plasmid, PRe-RDL also addresses the major limitation of RDL in that it abolishes any restriction on the gene sequence that can be oligomerized. The reconstitution of a functional plasmid only upon successful ligation in PRe-RDL also addresses two other limitations of RDL: the significant background from self-ligation of the vector observed in RDL, and the decreased efficiency of ligation due to nonproductive circularization of the insert. PRe-RDL can also be used to assemble genes that encode different sequences in a predetermined order to encode block copolymers or append leader and trailer peptide sequences to the oligomerized gene.

Nanostructured functional films from engineered repeat proteins

“…We use protein design as a powerful tool to tune structure, stability and function of proteins for specific applications. Current technologies are such that we can readily produce large quantities of homogeneous and monodisperse protein material using bacterial recombinant expression systems [16,17]. Here, we present designs for self-assembling functional protein arrays.…”

Tailored surface design of biodegradable endovascular implants by functionalization of poly (L-lactide) with elastin-like proteins

“…As previously reported by McDaniel et al, these DNA fragments were subcloned into pET24a (+), amplified and combined to ELP-CS5-ELP and ELP-P15-ELP by recursive directional ligation for generation of 2 repeats of the sequence [ELP-CS5-(ELP) 2 -P15-(ELP) 2 -CS5-ELP] 2 called X 2 [30] . This method of recursive directional ligation was used again to encompass the RGD sequence between two X 2 sequences.…”

Protein Tectons in Synthetic Biology

“…Current methods are mainly used to access individual proteins for intentional and rational design (Rodriguez-Cabello et al 2011;van Hest and Tirrell 2001;Gomes et al 2012). Strategies such as concatemerization and recursive ligation techniques (McDaniel et al 2010;Meyer and Chilkoti 2002;Goeden-Wood et al 2002) allow for the creation of defined sequences, but are limited if libraries of diversified long sequences are required. Polymerase chain reaction (PCR) based amplification strategies (e.g.…”