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Closing the Circuit on Cloning Complex Constructs
 
Patrick C. H. Lo, Ph.D.
BioTechniques, Vol. 55, No. 6, December 2013, pp. 287–289
Full Text (PDF)

Mention the words “Gibson cloning” and many synthetic biologists light up. The Gibson isothermal assembly technique (1), which involves multiple DNA fragments overlapping at their ends being simultaneously joined by an exonuclease, a DNA polymerase, and a DNA ligase, has been a boon to the field.

In the Gibson method, the exonuclease chews back the ends of each fragment, leaving 3′ single-stranded overhangs. Fragments anneal to each other through their complementary overhangs, with the gaps filled in by DNA polymerase and the nicks sealed by DNA ligase.

Although Gibson cloning has made a name for itself with synthetic biologists around the globe for its ability to join together multiple fragments to form large natural genes or even small bacterial genomes, the technique has not been used to assemble numerous transcriptional units (TUs) in a modular fashion for artificial genetic circuits, a key synthetic biology application. But now, two papers (2, 3) have been published describing similar methods for the rapid, modular construction of large, complex gene circuits that demonstrate the scalability of the Gibson technique, launching a new wave of Gibson fans.

Not UNSpecific

“It became pretty clear that something like Gibson technology could be one of those disruptive technologies that really changes how we approach synthetic biology,” explained Ron Weiss, associate professor in the Department of Biological Engineering at MIT and senior author of the paper (2) published in July in the journal Nucleic Acids Research. However, people who used the Gibson method “realized that it's not trivial to take parts and then create circuits out of them” and that the results depended on the actual sequence of the parts, with some working well and others not.

Synthetic biologists need to use standardized, well-characterized elements such as promoters, ribosome binding sites, and terminators for the TUs in a genetic circuit, which presented another critical consideration for Joseph Torella, a graduate student in Pamela Silver's lab in the Department of Systems Biology at the Harvard Medical School and lead author of the other Gibson assembly article that recently appeared in Nucleic Acids Research (3). “The challenge was that if you tried to Gibson together several parts where they have promoters and terminators on each end and they all kind of look alike, you can't do it because you need unique homology regions to facilitate the assembly.”

To solve these problems, both groups placed algorithmically generated unique nucleotide sequences (UNSs) at the ends of the component TUs to be Gibson-assembled into a single construct. Systematic use of these UNSs allowed components containing repeated sequences such as standardized promoters or terminators to be joined in an ordered fashion and ensured consistency in the efficiency of end joining during Gibson assembly.



Rapid and modular

Excited by the apparent scalability of Gibson cloning but still cognizant of the difficulties of designing overhangs for each set of sequences involved, Weiss sought “to create a platform that would really simplify the whole construction process…make it as predictable as possible…for even people with limited experience to be able to build very large circuits.”

In the Weiss lab's approach, which was developed for mammalian gene circuits, 40 bp UNSs were each computationally designed to maximize self-annealing in the Gibson reaction but to minimize misannealing to other sequences and hairpin formation.

The authors first constructed each TU by selecting a promoter and a gene from a library of standard sequence-verified parts and then cloning the pair using Gateway recombination into an appropriate destination vector called a “position” vector. Each position vector contains a chromatin insulator sequence, a Gateway recombination cassette, and a polyadenylation sequence, which are all flanked by two different 40 bp UNSs that are flanked in turn by two I-SceI restriction sites. (I-SceI is an extremely rare base cutter whose sequence is not found in the human or mouse genomes.)

I-SceI digestion of each position vector releases a fragment containing one TU with two different UNwSs at its ends. Ordered assembly of these TU fragments is then achieved in a Gibson reaction where the UNS 1/2 fragment is joined to the UNS 2/3 fragment, which is then joined to the UNS 3/4 fragment, and so on, until the final UNS (N-1)/N fragment is joined using an adapter vector containing UNS N/X to the carrier vector containing UNS 1/X and E. coli propagation sequences.

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