Part:BBa_K5300041

PnuoA-mCherry-RBS-vapC-Ter-PnifH-vapB-PglnK-sgRNA-mCherry-tracrRNA-PnuoA-Cas12k-double Terminator

The CRISPRi system is available to the suicide circuit. We used the Cas12k protein, which binds reversibly to target sequences under the guidance of sgRNA. When external signals reach a certain threshold, Cas12k inhibits transcription of corresponding sequence. Cas12k lacks cleavage activity, so it physically blocks transcription without cutting the target DNA sequence. As the intracellular concentration of sgRNA decreases, the bound Cas12k protein detaches from the target sequence, allowing normal transcription to resume when continuous repression is no longer needed.

We also involved the endogenous toxin-antitoxin (TA) system of the chassis Sinorhizobium fredii CCBAU45436.

In this composite part, vapC encodes the toxin, and vapB encodes the antitoxin. The expression of these genes is regulated by the promoters nifH and glnK. Under high oxygen conditions, expression of vapB is suppressed, and under low nitrogen conditions, the CRISPRi system is activated to suppress expression of vapC. When Sinorhizobium escapes the symbiotic environment and enters a high-nitrogen, high-oxygen external environment, vapC will be expressed normally, while vapB’s expression is suppressed by the high-oxygen conditions. This leads to toxin levels exceeding the tolerance threshold of the antitoxin, ultimately triggering the self-destruction of Sinorhizobium. This design prevents uncontrolled growth of Sinorhizobium in external environments, ensuring the system's safety and precision.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 2013
  Illegal SpeI site found at 1272
  Illegal PstI site found at 513
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 2013
  Illegal NheI site found at 1005
  Illegal SpeI site found at 1272
  Illegal PstI site found at 513
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 2013
  Illegal BglII site found at 1915
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 2013
  Illegal SpeI site found at 1272
  Illegal PstI site found at 513
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 2013
  Illegal SpeI site found at 1272
  Illegal PstI site found at 513
  Illegal NgoMIV site found at 1394
  Illegal AgeI site found at 5
  Illegal AgeI site found at 1601
  Illegal AgeI site found at 3797
  
• 1000
  COMPATIBLE WITH RFC[1000]