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.

Splice Overhang Extension (SOE)

The SOE approach to assembly involves PCR amplification of the sequences of interest using primers that will add to both ends sequences of about 20 bp in length that are complimentary to the intended upstream and downstream sequences. After SOE amplification, a second round of PCR amplification is carried out in which the amplified sequences act as their own primers at the assembly points. The flanking primers for the assembled sequence may be used to increase the yield of PCR product. No restriction enzymes are required. This process may be repeated until all the parts in the full construct are assembled. If desired, the flanking primers at the beginning and terminus of the full construct may be designed so as to included Standard Assembly restriction sites. After the final round of SOE assembly by PCR, the full construct can be digested and inserted into the BioBrick plasmid of choice. It is, however, also possible to assembly through SOE PCR an insert and a vector.