Part:BBa_K4195124

J23100-B0030-array(targeting pirA/B)-B0015-J23106-B0034-aca2-B0015

Biology

This part was designed as an element of type I CRISPR/Cas system, in which the array contains several designed spacers to target pirA/B gene and repeats derived from Vibrio parahaemolyticus FOC_022 (1) that could be recognized and handled by Cas proteins.

Some bacteria can not only encode anti-CRISPR protein (Acr) to inactivate the CRISPR/Cas system, but also encode anti-CRISPR-associated protein (Aca), which is the repressor of acr operon. Aca2 is one of the Aca family discovered with various anti-CRISPRs (2). The pathogen of AHPND, the Vibrio parahaemolyticus that can secret toxin PirA and PirB may also harbor acr and aca2 in the genome (3).

Usage and design

The sequence of array (BBa_K4195023) and aca2 (BBa_K4195028) were linked by overlap-extension PCR (OE-PCR), the product of which after being digested was then assembled into vector pUC57-simple to construct this composite part.

Characterization

Overlap-extension PCR (OE-PCR) for constructing the composite part

The sequence of array and aca2 were linked by OE-PCR. Target bands (1056 bp) can be observed at the position around 1000 bp (Fig. 1).

Fig. 1 DNA gel electrophoresis of OE-PCR products for linking BBa_K4195023 and BBa_K4195028.

Identification

When constructing this circuit, colony PCR and gene sequencing were used to verify that the transformatants were correct. Target bands (1110 bp) can be observed at the position around 1000 bp(Fig. 2).

Fig. 2 DNA gel electrophoresis of the colony PCR products of BBa_K4195124_pUC57-simple.

Reference

1. P. Baliga, M. Shekar, M. N. Venugopal, Investigation of direct repeats, spacers and proteins associated with clustered regularly interspaced short palindromic repeat (CRISPR) system of Vibrio parahaemolyticus. Mol. Genet. Genomics 294, 253-262 (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 7
  Illegal NheI site found at 30
  Illegal NheI site found at 362
  Illegal NheI site found at 385
  Illegal NheI site found at 599
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]