Using their method, Weiss's group first demonstrated they could assemble as many as 11 TUs into a single vector. They next cloned a 7-TU gene circuit into mammalian cells and observed strong ligand-inducible expression from the circuit. Finally, they used the technique to hierarchically assemble a 45 kb 7-TU module with 5 additional TUs to create a 63 kb 12-TU circuit. The additional TUs contained components already present in the 7-TU circuit, demonstrating that the assembly method was not affected by repetitive sequences in the assembled parts.Modular and rapid
For Torella, the impetus for improving Gibson assembly was his interest in optimizing fatty acid biosynthesis in bacteria. To accomplish this, he needed to co-express various proteins as well as build the necessary gene circuits.
As with Weiss's approach, Torella's method depends on 40-nucleotide UNSs placed at the ends of the genetic circuit components for their ordered and simultaneous Gibson assembly. In this case, 100,000 random 40-mer sequences were algorithmically screened to eliminate sequences that contained restriction sites or start codons, were GC-rich, formed hairpins, had high BLAST scores for the E. coli genome, and finally those that were likely to misassemble with each other. This screen left ~50 UNSs, of which 10 are described in the paper.
These UNSs were incorporated into “part” and “destination” vectors used for Gibson assembly of TUs into genetic circuits. Each part vector consists of a UNS “UN”, a multiple cloning site (MCS; with BioBrick and BglBrick sites), another UNS “UN+1”, and finally a third UNS “UX” that is common to all the part vectors. A promoter, a gene, and a terminator were cloned into the MCS of a part vector to create each TU of the final genetic circuit to be assembled. Prior to Gibson assembly, each TU construct was digested with restriction enzymes flanking UN and UN+1, except for the final piece, which was instead digested at restriction sites flanking UN+1 and UX. The gel-isolated, UNS-flanked TU fragments were then Gibson-assembled with a digested PCR-purified destination vector containing U1 and UX, with the order of the TUs determined by the UNSs flanking them.
It was clear that the presence of repeated sequences in the pieces had no effect on the success of assembly since they were able to efficiently assemble 5 pieces, each consisting only of a T7 promoter, MCS, and T7 terminator and which were 80% similar to each other, into a destination vector. The authors also demonstrated that strongly insulated parts could be assembled into expression libraries where the expression level of each TU was determined by its own promoter's strength without affecting expression levels of other TUs.
The authors used the method to construct and optimize a bacterial biosynthetic pathway that produces the chemical deoxychromoviridans using three genes from Chromobacterium violaceum. Each gene was combined with 6 possible promoters and 1 terminator sequence, and then all 216 combinations of the 3 genes were assembled and transformed into E. coli.
Testing of insert sizes and sequencing of randomly selected clones showed very efficient assembly, and these clones produced varying levels of deoxychromoviridans. Interestingly, the high-producing strains all had low expression of one gene and moderate expression of the two other genes, indicating that engineering strong expression of all three genes was not the optimal design strategy for this 3-TU biosynthetic pathway circuit, as might have been expected.
Finally, the authors used the technique to construct mammalian circuits by assembling two- and three-input split TALE AND gates into a single BAC for integration into mouse embryonic stem cells (ESC). Ironically enough, this wasn't an original goal of Torella's, and the results (though not the methodology) of the ESC work were actually published before his paper describing the technique. After his preliminary success using the method for assembling bacterial circuits, other groups in the Silver lab began using it as well for their projects.
“The mammalian cell people got so excited about it that they beat me to the punch and published their embryonic stem cell circuit work before I even published the methodology,” noted Torella with much amusement. Due to another paper on his fatty acid biosynthesis work that had some very demanding reviewers, “I got sidetracked for probably three solid months where I wasn't working on this assembly paper because of that,” explained Torella.