Amplified insert assembly: an optimized approach to standard assembly of BioBrickTM genetic circuits

The methods used for controlled modification of genetic material have experienced several major improvements since the initial development of recombinant DNA techniques in the early 1970s [1-4]. While there have been many efforts to simplify and standardize the genetic assembly process [5-7], one format that has gained widespread acceptance is the BioBrick？[8]. BioBrick assembly is commonly applied within a synthetic biology conceptual framework that abstracts and classifies basic functional units of genetic material (promoters, ribosome binding sites, protein coding sequences like reporters, terminators, etc.) as "parts" that can be assembled into "devices" that create new functionality, and higher level "systems" to accomplish complex tasks [9]. While the BioBrick standard has been revised several times [10-12], the basic format has remained the same. Each "BioBrick part" is contained on a plasmid and is flanked by four unique restriction sites, (two 5' and two 3') with the inner restriction sites that can be cut by two endonucleases yielding compatible ends. Using this format, individual parts may be selectively digested and then ligated together to form new composite parts or devices while preserving the format, connecting the individual parts by a benign mixed restriction site known as a "scar" (Figure 1). The chief utility of this technology is that all parts using the same format are interchangeable and composite parts can be recombined in the same way as individual parts [8]. This has allowed entire catalogs of compatible biological parts to be developed [9,13]. The major limitation of this format is that composite parts must be assembled piece by piece. To make this system useful and affordable, easy and reliable standard methods of performing these assemblies have been developed [14].The original method used to perform "bio-bricking" is called the standard assembly method. In using this method, one part is designated the insert and is digested out of its p