Part:BBa_K4190009

Golden Gate Assembly Flagged Backbone Primer Reverse for pET28:GFP

Flagged primer to add BsaI site to pET28 for Golden Gate Assembly.

Usage and Biology

Golden Gate Assembly (GGA) is a modular assembly method by which multiple DNA fragments can be simultaneously integrated into a plasmid backbone [1]. We designed gene fragments for both E. coli and S. cerevisiae to be used in GGA plasmid integration. GGA exploits type IIS restriction enzymes, which cleave DNA surrounding the recognition site, so the final recombinant product has no indication of integration. All type II restriction enzymes leave “sticky ends” on the backbone and the gene insert. These “sticky ends” bind to one another since they are designed to have complementary nucleotide sequences. What makes Type IIS restriction enzymes optimal for GGA is that they cut downstream of the enzyme binding site by a certain number of nonspecific nucleotides. This number varies based on the type IIS restriction enzyme used. This means that once the sticky ends can be engineered to seamlessly integrate into DNA without inserting residual nucleotides from the restriction enzyme binding site. This is termed “scarless integration” and prevents mutagenesis during transcription of the amino acid sequences. The deletion of restriction enzyme sites also means that digestion and ligation can be accomplished simultaneously.

For both E. coli and S. cerevisiae, we used the type IIS restriction enzyme, BsaI, on our insert. This restriction enzyme binds to the sequence GGTCTC and cuts after the first and fifth non-specific nucleotides, which results in “sticky ends” downstream of the binding site [2]. The restriction enzyme site was present on the top and bottom strands respectively in the five prime to three prime direction. The desired insert sequence was constructed to allow scarless integration into pET:28-GFP. Our flagged primers will add the restriction sites to our plasmid backbone through inverse pcr amplification, while simultaneously "cutting out' the GFP gene.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 8
  

References

[1] C. Engler, R. Gruetzner, R. Kandzia, and S. Marillonnet, “Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes,” PLoS ONE, vol. 4, no. 5, p. e5553, May 2009, doi: 10.1371/journal. pone.0005553.

[2] J. H. Lee, H. J. Won, E.-S. Oh, M.-H. Oh, and J. H. Jung, “Golden Gate Cloning- Compatible DNA Replicon/2A-Mediated Polycistronic Vectors for Plants,” Front. Plant Sci., vol. 11, p. 559365, Oct. 2020, doi: 10.3389/fpls.2020.559365.