Part:BBa_K2761010

Inducible message generator (RT/msr-msd)

The msr-msd sequence in the retron cassette is flanked by two inverted repeats. Once transcribed, the msr-msd RNA folds into a secondary structure guided by the base pairing of the inverted repeats and the msr-msd sequence. The RT recognizes this secondary structure and uses a conserved guanosine residue in the msr as a priming site to reverse transcribe the msd sequence and produce a hybrid RNA-ssDNA molecule called msDNA (i.e., multicopy single-stranded DNA).

The construct codes a SCRIBE system adaptation. This system generates single-stranded DNA inside of living cells in response to gene regulatory signals.

It is composed of 3 main components: A retrotranscriptase protein One msr RNA moiety which acts as a primer for the RT. And, another RNA moiety, called msd, which represents a template for the reverse transcriptase. This sequence contains the message of interest.

This part is an improvement of the BBa_K1681001 from UIUC_Illinois, instead of using a Recombinase we used CRISPR Cas 1-2 for inserting DNA sequences into the genome as a recorder.

Figure 1: Diagram of expression of the Retrotranscriptase and the msdDNA(DNA and RNA hybrid). Where de msr is the dark green segment and the msd is the light green segment, where the red segment represents the specific message that Cas1Cas2 complex recognizes to be integrated into the genome of the bacteria

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 435
  Illegal BamHI site found at 1355
  Illegal BamHI site found at 1419
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]