Coding

Part:BBa_K3894024

Designed by: Jiaqi Zhang   Group: iGEM21_NEFU_China   (2021-10-17)


Nb.BbvCI-R2-Phi29
The iGEM 2021NEFU_China designed Phi29（BBa_K3894014） to connect Nb.BbvCI-R2（BBa_K3894018）, and achieved the connection between Phi29 and ZF（BBa_K3894016） through the assembly of the BbvCI-R1（BBa_K3894017） and R2 subunits.

Description

The restriction enzyme Nb.BbvCI cleaves duplex DNA within a seven base-pair asymmetric recognition sequence. We designed Phi29（BBa_K3894014） to connect Nb.BbvCI-R2（BBa_K3894018）, and achieved the connection between Phi29 and ZF（BBa_K3894016） through the assembly of the BbvCI-R1（BBa_K3894017） and R2 subunits, to replace Phi29-slig and establish efficient protein interaction. Meanwhile, we mutated 3 nucleotides of the Nb.BbvCI-R2 subunit to inactivate its catalytic site. As a result, the two combined subunits had a single-strand cleavage activity, resembling that of nCas9.

Experience

The iGEM 2021 NEFU_China successfully induced and purified the recombinant Nb.BbvCI-R2-Phi29 protein, and the results are as follows:
Figure 1. SDS-PAGE analysis of purified recombinant Nb.BbvCI-R2-Phi29. |1 and 2. Elutent of His×6-tagged Nb.BbvCI-R2-Phi29 from Ni-NTA agarose beads.


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]


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Parameters
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