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Generating quick constructs for intracellular vesicular transport studies

A gBlocks® Gene Fragments application

IDT gBlocks® Gene Fragments provide a tool for easy gene construction or modification. Single gBlocks Gene Fragments can be used to introduce multiple modifications in a sequence [1] or substitute as a gene (e.g., for short genes such as an immunoglobulin (Ig) domain; see A Next Generation Understanding of Immune Response at www.idtdna.com/decoded). Several of these double-stranded DNA fragments of up to 3000 bp each can be quickly assembled using the Gibson Assembly™ method [2] to generate genes or other constructs. Thus you can re-create the elusive or damaged gene from your clone library, re-constitute a gene from an organism that is not readily accessible, or design and build a new function or chimera. Additional applications of gBlocks Gene Fragments include using them as template or sub¬strates for enzymatic reactions (polymerases, methylases, etc.) or as standards for qPCR [3].

Gene construction for protein engineering

In one example, the Kirill Alexandrov laboratory at the Institute of Molecular Biology (IMB), University of Queensland (Brisbane, Australia) uses gBlocks Gene Fragments in their studies of intracellular vesicular transport and the role played by Rab GTPases in docking and fusion of intracellular membranes. The lab performs a lot of protein engineering and gBlocks Gene Fragments have provided them with rapid and cheap access to new types of functional and structural elements that they do not possess in their plasmid database. Thus, the researchers can construct novel biological modules and cascades to better understand the tethering complexes and transcription-regulating complexes critical to their system.

Easy assembly by molecular biology novices

Scientists using gBlocks Gene Fragments use a variety of cloning methods to assemble the double-stranded, overlapping segments of DNA. For an overview of some of the cloning methods compatible with gBlocks Gene Fragments, see the Related DECODED Reading selection below.

The Alexandrov lab predominantly employs the Gibson Assembly method. They find it a very fast and robust technique that requires a minimal amount of skill and expertise. Dr Viktor Stein, a postdoctoral fellow in the lab (Figure 1), notes, “Students who are only in their third week in the lab are routinely assembling 3–4 DNA fragments right after they have mastered plasmid preps and PCRs.”

Figure 1. Dr Viktor Stein (left) and IDT Sales Representative, Keith Miller (right).

Fidelity of constructs

“The fidelity of gBlocks fragments is generally very good, especially for assembling synthetic DNA fragments less than 1 kb,” says Dr Stein. The group verifies cloned DNA assemblies by sequencing. Note that when assembling gBlocks Gene Fragments, IDT scientists recommend sequencing twice as many clones as the number of gBlocks fragments assembled (e.g., when assembling 3 fragments, you would sequence 6 clones).

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

References

  1. Bolisetty MT, Beemon KL. (2012) Splicing of internal large exons is defined by novel cis-acting sequence elements. Nucleic Acids Res, 40(18):1–11.
  2. Gibson DG, Young L, et al. (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods, 6(5):343–345.
  3. Yoon OK, Hsu TY, et al. (2012) Genetics and regulatory impact of alternative polyadenylation in human B-lymphoblastoid cells. PLoS Genet, 8(8):e1002882.

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

These selected articles discuss some of the cloning methods that are compatible with our custom dsDNA fragments, gBlocks® Gene Fragments.

Isothermal assembly: Quick, easy gene construction
Learn how, in a single reaction, isothermal assembly can combine several overlapping DNA fragments to produce a ligated plasmid ready for transformation.

Cloning strategies Part 1: Assembly PCR for novel gene synthesisLearn how you can use single-stranded oligos or a mix of single- and double-stranded DNA to produce longer genes of up to several thousand base pairs. No restriction sites are needed, and the approach is beneficial for assembling constructs that contain modular elements, such as antibodies. 
Cloning strategies Part 2: Cohesive-end cloning
Cohesive-end cloning is one of the most commonly employed techniques in molecular biology. Review these tips and tricks for cloning using restriction enzymes. 

Cloning strategies Part 3: Blunt-end cloning
Blunt-end cloning is one of the easiest and most versatile methods for cloning dsDNA into plasmid vectors. It is easy because the blunt-ended insert requires little to no preparation. Read an overview of blunt-end cloning with tips for making this cloning approach successful. 


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.


Author: Nicola Brookman-Amissah, PhD, is a senior scientific writer at IDT.

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