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 500 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 Glycon 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.

Related DECODED Reading

These selected articles from previous DECODED issues discuss just some of the cloning methods that are compatible with gBlocks® Gene Fragments.

Isothermal Assembly: Quick, Easy Gene Construction
This summary of the Gibson Assembly™ Method, developed by Dan Gibson, PhD, also provides tips on using this method to assemble gBlocks™ Gene Fragments.

Cloning Strategies Part 2: Cohesive-End Cloning
This is an overview of the well-established method for cloning double-stranded DNA using restriction enzymes that leave compatible ends for cohesive-end cloning. It also covers some plasmid basics.

Cloning Strategies Part 3: Blunt-End Cloning
This article describes blunt-end cloning of double-stranded DNA using T4 ligase, and includes some tips and tricks to increase experimental success.

Assembly PCR for Novel Gene Synthesis
Compatible with single- or double-stranded DNA starting material, including gBlocks Gene Fragments, assembly PCR is commonly used to produce genes of up to several thousand base pairs.