Cloning and subcloning have long been mainstay methods for molecular and cellular biologists. Time-to-results and biological accuracy of the cloned product have become important factors in designing cloning procedures as the edge of scientific inquiry advances beyond simple questions of gene expression and function.
Traditional cloning with restriction enzymes comes with some limitations. A vector’s structure may negatively affect downstream applications, including transferability between hosts and levels of protein expression, which can result in low efficiency. Success with restriction enzymes is dependent on the availability of specific sequences in the multiple cloning site, and those must not be present in the gene itself. Cloning with restriction enzymes is labor intensive and can take days to complete. Lastly, the traditional method typically leaves undesired recombination sequences of around 10 amino acids in the final expressed protein. These unnecessary sequences are referred to as “seams” or “scars,” and can significantly affect the biology of the gene product.
Seamless cloning methods
Researchers have developed a number of new strategies to overcome those limitations that are collectively referred to as seamless cloning.
These methods all use innovative techniques to insert a DNA segment into a plasmid without the need for restriction enzymes or prolonged, multistep protocols. For example, in Gibson assembly, which was created by Daniel G. Gibson in collaboration with the J. Craig Venter Institute, plasmids and primers are designed with two identical sequences of about 40 base pairs on each end. An exonuclease digests one strand of DNA back from each 5’ end, creating a single-stranded region that can anneal to its complementary sequence on the vector or plasmid. DNA polymerase is used to close the gaps, and DNA ligase links the joined segments together to create continuous sequence. The entire process is carried out in a single, isothermal reaction.1
Seamless cloning has been used for all of the same applications as traditional restriction enzyme-based cloning, and beyond.
Golden Gate Assembly by New England Biolabs allows the insertion of multiple gene inserts into a vector using a type IIS restriction enzyme, which cuts outside of its recognition sequence, and T4 DNA ligase. When the cleavage sites are designed correctly, the plasmid is assembled without the original restriction site.2
Agilent SureVector is a modular vector assembly system built on the synthetic biology concepts of standardized parts. Agilent selected the most popular vector components and made them into functional modules, including promoters, tags, selectable markers, and origins of replication. In the SureVector system, there is no vector backbone. Instead, the individual modules are assembled to build the vector. “Researchers do not have to spend time designing overlaps, as required with other methods to maintain functionality, or selecting sites as required with restriction enzyme cloning,” says Laura Whitman, global product manager for Agilent Technologies.
In vivo methods
Methods like Gibson assembly and Golden Gate Assembly have been around since the late 1990s, but researchers have continued to pursue innovative seamless cloning methods. In recent years, a number of those newer methods have been published.
AQUA (advanced quick assembly) cloning, takes the cloning reaction into a living system by taking advantage of intrinsic in vivo processing of linear DNA fragments by E. coli.3 SLiCE (seamless ligation cloning extract) is based on bacterial extracts from certain strains of E. coli modified to optimize cloning efficiency. The bacterial strains are deficient in RecA, a DNA repair protein, and the cloning is carried out with vector and insert DNA fragments that have short end homologies. The process is done in a single tube and takes one hour, followed by standard transformation into host bacteria.4
Finding a common starting point
Many cloning systems, especially those that are commercially marketed, make impressive claims for total cloning times such as one hour from start to finish. However, according to Pawel Jajesniak, a researcher with the ChELSI Institute and Advanced Biomanufacturing Center at the University of Sheffield, those claims often don’t include a common starting point. “Many cloning methods in their description or when advertised assume that one starts with two linearized DNA fragments, often with complementary terminal regions already in place, which is rarely the case in during normal experiments,” Jajesniak says. For example, according to one vendor, 15–60 minutes is needed for its single-tube assembly reaction. However, Jajesniak points out that the researcher first needs to isolate the desired DNA fragment—most likely amplifying it by PCR—and linearize the cloning vector—likely through a second PCR. Overlapping ends must also be added to one of the DNA fragments.
“Only then, the two fragments can be joined together by performing the 15–60 minute single tube reaction provided by Gibson Assembly Master Mix. As a result, the total cloning time needed includes both the time of the two PCRs and the time of the assembly reaction,” says Jajesniak.
Megaprimer-based methods
Jajesniak developed a method using megaprimer-based PCR of the whole plasmid that does not include pre-assembly preparatory steps. In the first stage of megaprimer-based cloning, the DNA fragment of interest is amplified during PCR using specifically designed primers, which are part of the oligonucleotide sequence corresponding to an amplified fragment and the recipient plasmid. As a result, the amplified fragment contains terminal DNA regions complementary to the annealing sites on the recipient plasmid. That fragment is purified and used as a megaprimer for the second PCR, in which the desired recombinant vector is created.5
“The strong point of megaprimer-based methods is that they attempt to incorporate DNA assembly into a PCR that is already required for many experiments involving sequence-independent cloning—that is to linearize the recipient vector,” says Jajesniak.
Applications
Seamless cloning has been used for all of the same applications as traditional restriction enzyme-based cloning, and beyond. Some options for applications of seamless cloning include protein expression optimization and shotgun cloning.
In protein expression optimization, a seamless cloning technology is used to test the effects of multiple promoters and tags on protein expression levels. “Traditionally, researchers would try to express their gene from a single vector. If it didn’t express well, they might try a different vector or promoter, or add a solubility tag. However, doing this on a large scale can be expensive, time-consuming and difficult,” says Whitman. Agilent demonstrated that protein expression optimization can be carried out in a single reaction using Agilent’s SureVector system. Agilent tested three unique promoters and six unique tags in one reaction, then screened for expression of all 18 possible promoter-tag expression combinations. According to Whitman, “Different promoter-tag combinations had vastly different expression levels unique to each gene.”
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Shotgun cloning is a high-throughput cloning application using multiple DNA fragments. The fragments with the same homologous ends are used for Gibson assembly cloning with a single vector.
Seamless cloning and other synthetic biology applications are gradually replacing old school cloning with restriction enzymes. Researchers are still using older cloning methods for some applications, and those methods are reliable and inexpensive options. However, seamless cloning can significantly streamline the cloning process, reducing a week of work to potentially one hour. Moreover, the advantages of a “seamless” gene product offer more faithful replication of the biology of the original gene product, which can be critical in some studies.
References
1. Gibson, DG, Young, L, Chuang, R, Venter, C, Hutchison, CA, Smith, HO, “Enzymatic assembly of DNA molecules up to several hundred kilobases,” Nature Methods, 6, 343 - 345, 2009. [PMID: 19363495]
2. Engler, C, Kandzia, R, Marillonnet, S, PLoS One, 3(11): 3647, “A One Pot, One Step, Precision Cloning Method with High Throughput Capability,” PLoS One, 3(11):e3547, 2008. [PMID: 18985154]
3. Beyer, HM, Gonschorek, P, Samodelov, SL, Meier, M, Weber, W, Zurbriggen, MD, “AQUA Cloning: A Versatile and Simple Enzyme-Free Cloning Approach,” PLoS One, 10(9):e0137652, 2015. [PMID: 26360249]
4. Zhang Y, Werling U, Edelmann, W, “SLiCE: a novel bacterial cell extract-based DNA cloning method,” Nucleic Acids Res, 40(8):e55, 2012. [PMID: 22241772]
5. Jajesniak, P, Wong, TS, “QuickStep-Cloning: a sequence-independent, ligation-free method for rapid construction of recombinant plasmids,” J Biol Eng, 9:15, 2015. [PMID: 26388935]
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