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Related Experiment Video

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Use of In Vivo Assembly for High-efficiency Plasmid Construction
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Seamless Insert-Plasmid Assembly at High Efficiency and Low Cost.

Roger M Benoit1, Christian Ostermeier2, Martin Geiser3

  • 1Laboratory of Biomolecular Research, Paul Scherrer Institut, Villigen, Switzerland.

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|April 14, 2016
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Summary

Improving seamless cloning, this study found that filling single-stranded gaps in plasmids boosts transformation efficiency. A modified Gibson assembly using T5 exonuclease and Phusion DNA polymerase offers a cost-effective and robust method for precise DNA assembly.

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Area of Science:

  • Molecular Biology
  • Biotechnology
  • Synthetic Biology

Background:

  • Seamless cloning techniques like Gibson assembly are crucial for plasmid construction.
  • Existing methods face challenges such as low efficiency (co-transformation) or high cost (Gibson assembly).
  • Optimizing these methods is vital for advancing genetic engineering and synthetic biology.

Purpose of the Study:

  • To enhance the robustness and reduce the cost of seamless cloning experiments.
  • To investigate the role of single-stranded DNA ends in co-transformation cloning.
  • To determine the impact of single-stranded gaps in plasmids on sequence and ligation-independent cloning (SLIC) efficiency.

Main Methods:

  • Examined the importance of complementary single-stranded DNA ends for co-transformation cloning.
  • Assessed the influence of single-stranded gaps in circular plasmids on SLIC cloning efficiency.
  • Utilized DNA polymerase to fill single-stranded gaps and T5 exonuclease with Phusion DNA polymerase for a modified Gibson assembly.

Main Results:

  • Single-stranded gaps in double-stranded plasmids significantly decrease transformation efficiency in E. coli.
  • Filling these gaps with DNA polymerase markedly increased transformation efficiency.
  • A modified Gibson assembly using only T5 exonuclease and Phusion DNA polymerase, omitting Taq DNA ligase, proved highly efficient and cost-effective.
  • Successful assembly was achieved with short, 15-nucleotide overlap regions.

Conclusions:

  • Addressing single-stranded gaps is critical for improving the efficiency of seamless cloning techniques like SLIC.
  • A simplified and cost-effective Gibson assembly protocol can be achieved by omitting Taq DNA ligase.
  • This optimized method allows for highly efficient DNA assembly with minimal overlap sequences, reducing costs and enhancing experimental robustness.