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Fast and accurate assembly of a 2.1 kb gene

gBlocks® Gene Fragments + Gibson Assembly® Master Mix

New England Biolabs (NEB) is a manufacturerof high quality molecular biology reagents, including restriction and modification enzymes, for genomics research. In fact, much of our knowledge about these enzymes has come from basic research performed by scientists in laboratories at NEB. 

Over the past decade, Next Generation Sequencing (NGS) has provided molecular biologists, enzymologists, and life science technology innovators unprecedented access to large collections of interesting candidate enzymes for research and technology development. In order to characterize the activity of these potentially useful enzymes, researchers must clone the gene of interest and express the protein in vitro or in vivo. NEB uses a variety of approaches to clone these genes, including traditional restriction enzyme cloning, USER™ cloning, as well as a number of newer DNA assembly approaches (e.g., assembly of single-stranded synthetic oligonucleotides).

Rapid cloning using isothermal assembly

The development of gBlocks Gene Fragments by Integrated DNA Technologies, Inc. (IDT) has reduced the time, error rate, and costs associated with producing single gene constructs or multiple genes within metabolic pathways. For NEB, use of gBlocks Gene Fragments and the recently introduced NEB Gibson Assembly® Master Mix has reduced both the hands-on time and the total turnaround time of some of their cloning workflows.

For example, in connection with an internal research project, NEB researchers expedited the cloning of a 2.1 kb gene into a linearized pET21-a bacterial expression vector using 5 gBlocks Gene Fragments that spanned the entire 2.1 kb sequence. Each gBlocks fragment included a 20 bp overlap between adjacent fragments and a 20 bp overlap between the free ends of the vector and the gene assembly (see Figure 1). Gene fragments were quickly assembled with the linearized vector to create a 7.5 kb gene construct using the Gibson Assembly Master Mix and associated protocol. 

Figure 1. Cloning of 2.1 kb gene into pET21-a plasmid using IDT gBlocks® Gene Fragments and Gibson Assembly® Master Mix. To produce a 2.1 kb gene, 5 gBlocks Gene Fragments spanning the gene sequence were ordered, each with 20 bp of overlap with the adjacent fragment. The 3 enzymes in the Gibson Assembly Master Mix (NEB) work together to assemble the individual DNA fragments, in 1 hr, 50°C. A 5’ exonuclease chews back DNA ends leaving overlapping 3’ ends (orange), which then anneal. A high fidelity DNA polymerase fills in double-stranded gaps generated by the exonuclease. In the final step, a DNA ligase joins individual fragments [1]. Following assembly, 10 transformant colonies were analyzed, resulting in 2 plasmids with the correct sequence. Cloning and expression of this 2.1 kb construct were completed in 4 days.

Assembly protocol

To assemble the gene clone from 6 fragments (5 gBlocks Gene Fragments and the vector), the lab used 20 ng (0.06 pmol) of each gBlocks Gene Fragment and 0.05 pmol of linearized vector. (IDT supplies 200 ng (0.6 pmol) of each gBlocks Gene Fragment). Assembly was straightforward—the appropriate amounts of gene fragment and vector DNA were added to the Gibson Assembly Master Mix (NEB #E2611) and incubated at 50°C for 1 hour, followed by transformation into T7 Express Competent E. coli (NEB #C2566). Transformant colonies (n=10) were picked, cultured overnight, and plasmid DNA prepared from each transformant. DNA from each colony was sequenced to confirm the integrity of the entire 2.1 kb region. 

Figure 2. Estimated timeline for cloning with gBlocks® Gene Fragments and Gibson Assembly® Master Mix.

Clone analysis

In this experiment, 2 of the 10 colonies picked carried the correct 2.1 kb DNA sequence. The expressed protein was then confirmed by SDS-PAGE and further characterized using a biochemical assay. Cloning, sequencing, and characterization of protein activity took less than 4 days, shorter than necessary for a third party to synthesize the complete gene and provide a finished construct. An estimated timeline for Gibson Assembly cloning using gBlocks Gene Fragments is shown in Figure 2.

The use of gBlocks fragments to construct this 2.1 kb gene and assemble it into the pET21-a expression vector was simple and cost-effective, saved time, and resulted in fewer errors than the assembly of DNAs from single-stranded synthetic oligonucleotides. These benefits ensure that Gibson Assembly cloning using gBlocks Gene Fragments will be a method NEB researchers will continue to work with in the future.

Read a description of the Gibson Assembly® method in the DECODED newsletter article, Assembling gene fragments using isothermal assembly.


References

  1. Gibson DG, Young L, et al. (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods, 6(5):343–345.

Product focus—custom dsDNA fragments

gBlocks® Gene Fragments

These double-stranded, sequence-verified, DNA genomic blocks, 125–3000 bp in length, are designed by you, and are shipped in 2–5 working days for affordable and easy gene construction or modification. They have been used in a wide range of applications including CRISPR-mediated genome editing, antibody research, codon optimization, mutagenesis, CRISPR genome editing, and aptamer expression. They can also be used for generating qPCR standards.


gBlocks Gene Fragments Libraries

gBlocks Gene Fragments are also available as dsDNA fragment pools that contain up to 18 consecutive variable bases (N or K) for recombinant antibody generation or protein engineering.


Learn more about gBlocks Gene Fragments at www.idtdna.com/gblocks.

Related reading

Longer gBlocks® Gene Fragments Make Gene Assembly Simple—Learn how you can use made-to-order, double-stranded DNA fragments up to 3 kb to simplify your cloning and mutagenesis protocols.

Towards next generation biosensors—Research profile: Read how fluorescent protein-based Ca2+ indicators based on naturally occurring substrates are assembled using gBlocks Gene Fragments. These sensors are being developed to monitor in vivo neural activity.

Benefits of codon optimization—Planning to express a gene in an heterologous system? Learn how rebalancing codon usage is important for optimizing protein expression. While there are no known methods to predict protein expression, as numerous factors contribute to ultimate protein yield, codon optimization plays a critical role.

Codon optimization tool makes synthetic gene design easy—Use the free IDT Codon Optimization Tool to simplify designing synthetic genes and gBlocks® Gene Fragments for expression in a variety of organisms. The tool allows for manual changes, and takes into account natural codon bias and synthesis complexity.


Review other DECODED Online newsletter articles on gBlocks Gene Fragment use in synthetic biology applications.


You can also browse our DECODED Online newsletter for additional application reviews, lab tips, and citation summaries to facilitate your research.


Authors: Peichung Hsieh, PhD is an application scientist at New England Biolabs, Inc and Hans Packer is a scientific writer at IDT.

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