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Generating quick constructs for intracellular vesicular transport studies

Research profile: gBlocks Gene Fragments can provide rapid and cheap access to new types of functional and structural elements. See how these researchers use the custom dsDNA fragments for construction of novel biological modules and cascades to better understand the tethering complexes and transcription-regulating complexes critical to intracellular vesicular transport.

Jan 18, 2013

IDT gBlocks® Gene Fragments provide a tool for easy gene construction or modification. Single fragments can be used to introduce multiple modifications into a sequence, or as genes substitutes (e.g., for short genes such as an immunoglobulin (Ig) domain) [1–2]. Several of these double-stranded DNA fragments of up to 3000 bp each can be quickly assembled using the Gibson Assembly™ method to generate genes or other constructs [3]. 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 substrates for enzymatic reactions (polymerases, methylases, etc.) or as standards for qPCR [4].

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

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.”

YR-Victor Stein

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).

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. Packer H. (2012) A next generation understanding of immune response. DECODED Online Newsletter, IDT.
  3. Gibson DG, Young L, et al. (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods, 6(5):343–345.
  4. Yoon OK, Hsu TY, et al. (2012) Genetics and regulatory impact of alternative polyadenylation in human B-lymphoblastoid cells. PLoS Genet, 8(8):e1002882.

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