| Popis: |
The Golden Gate technique allows the assembly of genetic sequences from libraries of standardized basic components, which are cleaved from their donor vectors and concatenated in the acceptor vector in a defined order. This reaction is subsequently transformed into competent bacterial cells that are grown overnight to reveal positive colonies carrying the correctly assembled vector. The cleavage of the components is done by a TypeIIS restriction enzyme, which recognizes asymmetric DNA sequences and cleaves outside of this site, leaving overhangs in a directional way (Figure 1A). This property allows the creation of ordered assembly positions by defining synthetic syntaxes of these overhangs (Figure 1B). Within the different methodologies and libraries based on Golden Gate, some methods, such as uLoop Assembly (Pollak et al, 2020), allow the cyclic assembly of increasingly large and complex vectors in which the products of a reaction are the substrates of the next level reactions (Figure 2). By this means it is possible to build libraries of easily reusable components to perform assemblies in a combinatorial way of odd and even assembly levels. The standardized DNA components have to be flanked by the enzyme recognition sites and the proper syntax according to the desired position of assembly. These DNA pieces can be obtained from previously created libraries or custom made. The reaction operates cycling between 37°C and 16°C. At the first temperature, the restriction enzyme cuts the DNA pieces. These pieces are concatenated by their overhangs sequence homology and sealed by the catalysis of a ligase enzyme at the second temperature. Once a piece is correctly assembled it cannot be cutted again, and cycle by cycle, the pieces are concatenated in the final assembly product. |
