Part:BBa_K3894025

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

Description

The iGEM 2021 NEFU_China designed ZF（BBa_K3894016） to connect BbvCI-R1（BBa_K3894017）, and achieved the connection between ZF and Phi29（BBa_K3894014） through the assembly of the Nb.BbvCI-R2（BBa_K3894017） and R1 subunits that could achieve anchoring and chain cleavage. Meanwhile, the binding of two subunits BbvCI-R1 and R2 to Phi29 and ZF was accomplished, which made the proteins efficiently assembled.

Experience

The iGEM 2021 NEFU_China successfully induced and purified the recombinant BbvCI-R1-ZF protein, and the results are as follows:

Figure 1. SDS-PAGE analysis of purified recombinant Nb.BbvCI-R1-ZF. The weight of ZF3-BbvC R1 protein is 50.6 kDa.|1 and 2. Elutent of His×6-tagged ZF-BbvCI-R1 from Ni-NTA agarose beads.

Figure 2. EMSA to compare the nickase activity of nCas9 and Nb.BbvCI. |1-4. verification of the nCas9 nickase activity. 5-8. verification of Nb.BbvCI nickase activity.

We respectively verified the deletion activity of nCas9 and Nb.BbvCI, both of which have the ability of deletion of double-stranded DNA. For further screening, we decided to compare their deletion activity under the same conditions. Through EMSA, we concluded that Nb.BbvCI had a stronger single chain deletion activity than nCas9, and we finally selected ZF3-BbvC R1 as our specific targeting tool.

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]