Part:BBa_K4630100

stgRNA-barcode-cassette 1

This part is the barcode-cassette form of the self-targeting guide RNA 1 (stgRNA 1). It is the building block of the cascade recording system. It contains Lac promoter, unique barcode, self-targeting guide RNA (stgRNA 1), double terminator and the barcode. It is used in the verification of the self-targeting and self-recombination system. The unique barcode is used to distinguish each cassette and provide unique homologous arm.

Background of the Cassette

Cassette recorder is the leading edge of DNA memory device. By changing cassettes, the entire recorder is capable to record many rounds of biological events. However, the method of DNA manipulation is the biological basis of the DNA-based recording. Perli et al. ’s work demonstrates that based on the CRISPR/Cas9 and self-targeting guide RNA (stgRNA), editing could happen in the stgRNA sequence¹ (fig 1a). Also, Zhao et al. ’s work enlightens us with the concept of intra-plasmid recombination² (fig 1b). So, we integrate the two methods into a single cassette. Once triggered, double strand break (DSB) is introduced into the working plasmid, enabling the intra-plasmid recombination. It is worth noting that the two homologous arms flank the stgRNA, so the entire cassette will be deleted after the recombination, and the next cassette is expected to be brought to the proper expressing place. The inducer triggers over and over, and recorder records it by keeping changing the cassette.

Figure. 1 DNA manipulating methods from the literatures

Usage and Biology

We integrate the two methods into a single cassette, with unique barcode (fig 2). Once triggered, double strand break (DSB) is introduced into the working plasmid, enabling the intra-plasmid recombination. It is worth noting that the two homologous arms flank the stgRNA, so the entire cassette will be deleted after the recombination, and the next cassette is expected to be brought to the proper expressing place. The inducer triggers over and over, and recorder records it by keeping changing the cassette.

Figure. 2 Working pattern of the change of cassette

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 55
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 94