Part:BBa_K3645011

Target-AID (CBE)

Contains the full CDS of Target-AID, whose Cas9 part was replace with our lab's dCas9.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1099
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 4775
  Illegal BamHI site found at 3378
  Illegal XhoI site found at 4384
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Contribution From XMU-China 2020

Group: iGEM Team NNU-China 2021

Author: Yan Xu

Summary: Testing its gene editing efficiency in BL21 (DE3)

Characterization from iGEM21-NNU-China

        MazF is toxin protein in MazF-MazE, toxin-antitoxin module. It was first registered in 2013 and used as an mRNA endonuclease. It kills bacteria without cracking them, which will directly influence the value of OD₆₀₀.

        In order to quantify the toxicity of mazF, colony forming unit (CFU) cell viability assays were used to measure functionality of the circuit(1).

        Because direct expression of MazF will kill bacteria, we construct the circuit "P_Bad/araC-RBS-MazF-terminator-pSB1C3"(BBa_K3332083) and "P_Bad/araC-RBS-EYFP-terminator-pSB1C3"(BBa_K3332082) in E.coli BL21(DE3) to characterize its function.

Fig.1 genetic circuit of P_Bad/araC-RBS-MazF-terminator.

        When 0.2% arabinose was added, the experimental group showed a sharp decrease on the CFU, which indicates the protein encoded by MazF is fatal to E. coli. It further demonstrated that the MazF encoded by J23100-RBS (BBa_K880005) was able to work as a toxin.

Fig.2 The results of CFU.The round dot indicates the non induction group, and the square dot indicates the induction group.