Part:BBa_K4195028

Biology

Aca2

Some bacteria encode anti-CRISPR protein (Acr) to inactivate the CRISPR-Cas system and can also express anti-CRISPR-associated protein (Aca), which is the repressor of acr operon (1). Aca2 is one of the Aca family discovered with various anti-CRISPRs (2). The pathogen of AHPND, the Vibrio parahaemolyticus that can secrete toxin PirA and PirB may also harbor acr and aca2 in the genome (3).

Usage and design

In order to prevent our CRISPR-system from being inhibited by the endogenous anti-CRISPR system in AHPND pathogenic Vibrio parahaemolyticus, we determined to choose the Aca2 to repress the expression of potential AcrIF proteins. We used both BBa_J23106 and BBa_B0034 to construct the expression circuit and obtained the composite part BBa_K4195129, which are assembled into the vector pUC57-simple by standard BioBrick assembly. The constructed plasmid was transformed into E. coli DH5α, then the positive transformants were selected by chloramphenicol and confirmed by colony PCR and sequencing.

Characterization

Identification

When constructing this circuit, colony PCR and gene sequencing were used to verify that the transformants were correct. Target bands (2655 bp) can be observed at the position between 2000 bp and 3000 bp (Fig. 1).

Fig. 1 DNA gel electrophoresis of the colony PCR products of BBa_K4195129_pUC57-simple.

Reference

1. S. Y. Stanley et al., Anti-CRISPR-Associated Proteins Are Crucial Repressors of Anti-CRISPR Transcription. Cell. 178, 1452-1464 (2019).
2. N. Birkholz, R. D. Fagerlund, L. M. Smith, S. A. Jackson, P. C. Fineran, The autoregulator Aca2 mediates anti-CRISPR repression. Nucleic Acids Res. 47, 9658-9665 (2019).
3. A. Pawluk et al., Inactivation of CRISPR-Cas systems by anti-CRISPR proteins in diverse bacterial species. Nat. Microbiol. 1, 16085 (2016).

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 183
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]