DNA Cloning Tips – Build Clones with DNA Fragments using Gibson Assembly

Building large DNA constructs that contain no extraneous sequences is often a challenging task. One of the key engineering tools designed to help in constructing these large constructs is Gibson Assembly cloning (1).

The Gibson Assembly method allows multiple overlapping DNA fragments to be seamlessly linked in a one-step, single-tube, isothermal reaction (Invitrogen GeneArt Gibson Assembly HiFi Cloning Kit), or a two-step reaction (GeneArt Gibson Assembly EX Cloning Kit). DNA fragments of different lengths are assembled using complementary overlaps between fragments (Figure 1). The flexibility of this approach permits the construction of small and large DNA constructs and works with both single and multiple inserts.

Gibson Assembly technology uses homologous recombination to assemble adjacent DNA fragments that share end-terminal homology. The optimal length of the homologous fragment ends region depends on the number and length of the fragments in the assembly reaction (Table 1).

How can you use Gibson Assembly when your DNA fragments do not share any homology?  The answer is to use stitching oligonucleotides. Stitching oligonucleotides work as a bridge between the DNA fragments to be joined by sharing half of the sequence with each fragment. When doing one- and two-fragment cloning, two or three stitching oligonucleotides can be used with the Gibson Assembly method. This approach offers flexibility and enables virtually any possible combination between DNA fragments (Figure 2). The technique can be used in several ways, including carrying out a cloning strategy without generating a new PCR amplicon, or cloning the same PCR amplicon in multiple vectors without repeating PCR amplification of the fragment.

This strategy is helpful when working with large (>10 kb) or complex DNA inserts with high GC content or secondary structures. In this case, simply cut the insert using restriction enzymes, purify, and then add to the assembly reaction with your vector and stitching oligonucleotides.

What is the best approach? Should you piece together smaller fragments or assemble larger fragments?

When assembling large DNA constructs, the best approach is to keep the number of fragments as low as possible in order to maximize cloning efficiency. The size of the fragments, however, also plays an important role—the larger the fragments, the more difficult it is to prepare them in the quantities and purities needed for Gibson Assembly cloning. Therefore, for optimal efficiency, use a fragment number that is as low as possible while still preserving the minimum quantity and purity needed by your reaction. It is therefore a balance to keep the number of fragments low and the size not too large such that the quantity and purity required is not negatively affected.

Another important consideration for your Gibson Assembly reaction is selecting a cloning vector. It is recommended to use low-copy plasmids as vectors when using E. coli as the host cell. The use of high-copy plasmids containing large DNA constructs will most likely result in E. coli using DNA repair mechanisms to shrink or trim your plasmid. If this occurs, parts of your insert could be eliminated to make room for the elements needed for growth (such as the replication origin and selective markers). To avoid host-mediated selection, always use low-copy plasmids to allow for propagation of the full-length construct without hampering the E. coli DNA replication cycle.

During transformation steps, bacterial host cells can be prepared to take up foreign DNA using of two methods: electroporation or chemical transformation. While either method is suitable, the use of electroporation with electrocompetent cells often provides higher transformation efficiencies, but the process will require an electroporator.
As such, your method of selection will primarily depend on your lab setup. Regardless of your choice, GeneArt Gibson Assembly HiFi and EX Cloning kits are available with both chemically and electrocompetent cells.

In Figure 3, the GeneArt Gibson Assembly EX Cloning Kit was used to build large DNA constructs in the low-copy pASE101 vector (2). The inserts and vector with 50 bp of homology at each end were amplified using Platinum SuperFi II DNA Polymerase and purified with a GeneJET Gel Extraction Kit. Two and four fragments were assembled into the pASE101 vector to build 25 kb and 50 kb inserts, respectively (Figure 3A and 3B). To maximize transformation efficiency, the assembly product was precipitated with 100% ethanol and resuspended in nuclease-free water. A 10 μL aliquot was then used to transform ElectroMAX DH10B electrocompetent cells. Sixteen colonies were analyzed to calculate cloning efficiency (Figure 3C). While cloning efficiency was low (between 6 and 20%), high colony numbers made it possible to identify a colony containing the full-length insert in both cases.

Gibson Assembly Cloning is a powerful and flexible cloning method. It uses homology to seamlessly combine fragments, but oligonucleotide stitching can also be used for fragments that do not share homology. The two-step method in the case of the GeneArt Gibson Assembly EX kit can be used to build large constructs (> 50 kb) and remains one of the key cloning methods in synthetic biology research.

1. Enzymatic assembly of DNA molecules up to several hundred kilobases. Gibson et. al., Nat Methods. 2009 May; 6(5):343-5.
2. Baek C-H, Chesnut J, and Katzen F (2015) Positive selection improves the efficiency of DNA assembly. Anal Biochem 476:1–4.

Gibson Assembly® is a registered trademark of SGI-DNA, Inc. used under permission and license.