Assembly

We used three different methods of assembly over the course of this project, the detailed methods and materials of which may be found on the Protocols page. Below is an overview of the different assembly methods used.

Golden Gate Assembly

Golden Gate Assembly involves amplifying out the DNA sequence of interest with forward and reverse primers that add to both ends a BsaI restriction site and a 4 bp overhang. The reverse 4 bp overhang is designed to be complimentary to the forward 4 bp overhang of the intended downstream sequence. Likewise, the forward 4 bp overhang is designed to be complimentary to the reverse 4 bp overhang of the intended upstream sequence.

After Golden Gate PCR amplification with the appropriate primers, the parts can be assembled in a one-pot reaction involving the BsaI restriction enzyme, T4 DNA ligase, T4 DNA ligase buffer, and BSA. The image included here illustrates the Golden Gate Assembly approach, where the 'nnnn' sequences indicate the 3' end of the upstream part and the 5' beginning of the downstream part, and the '1234' sequences indicate the arbitrary 4 bp overhangs that will be added through Golden Gate amplification.

The paper Generation of Families of Construct Variants Using Golden Gate Shuffling (Engler C., Marillonnet S., Methods in Molecular Biology Volume 729, 2011, pp 167-181) provides a detailed description of the Golden Gate assembly approach and its applications.