Part:BBa_K3894018

Nb.BbvCI-R2
The iGEM 2021 NEFU_China created the E177G mutant of R.BbvCI-R2. We used the R1⁺R2^- mutant, named as Nb.BbvCI, which can only cleaves the bottom chain.

Description

The iGEM 2021 NEFU_China created the E177G mutant of R.BbvCI-R2. We used the R1⁺R2^- mutant, named as Nb.BbvCI, which can only cleaves the bottom chain. Initially, we used the commercial Nb.BbvCI to verify our design. The recognition sequence of restriction endonuclease R.BbvCI from Bacillus brevis is CC^TCAGC/GC^TGAGG and the R2 subunit cleaves the top chain CC^TCAGC. Nb.BbvCI is used instead of nCas9(BBa_K3894015) for single chain digestion. This subunit contains a catalytic site for DNA strand hydrolysis, with independent action and strand specificity^[1]. We mutated 3 nucleotides of the R.BbvCI-R2 subunit to inactivate the catalytic domain and used the mutant to form dimeric enzymes. As a result, the combination of the two subunits would form an engineered enzyme with single-strand cleavage activity.

Experience

Through communicating with other teams, we decided to use electrophoretic mobility shift assay (EMSA) to verify the activity of ssDNA replacement. Due to the limitation of experimental time, we used commercial Nb.BbvCI for verification. Based on the template DNA sequence, we synthesized a FAM-labeled probe with 23-nucleotide in length, and an unlabeled complementary oligonucleotide, named as c-probe.

Figure 1. EMSA to verify the nickase activity of the Nb.BbvCI. |1. Without Nb.BbvCI. 2. With Nb.BbvCI. 3. FAM-probe alone. 4. Annealed FAM-probe/c-probe.

References

[1] DF Heiter, Lunnen K D , Wilson G G . Site-specific DNA-nicking mutants of the heterodimeric restriction endonuclease R.BbvCI.[J]. Journal of Molecular Biology, 2005, 348(3):631-640.

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
  COMPATIBLE WITH RFC[1000]