Part:BBa_K2785003

Multiple Cell Recording (2 Repeat)

Our designed system of chronological event recording consists of a CRISPR/Cas9 base editor with two sgRNAs controlled by separate inducible promoters and a recording plasmid containing repeating units of DNA. gRNA #1 directs the base editor to make a mutation on the recording plasmid at the current frame. Once that mutation is made, this allows gRNA #1 to recognize the next frame. By constantly shifting the frame that is able to record, the system is keeping track of elapsed time. Under the presence of a stimulus, gRNA #2 is produced and directs the base editor to mark the current frame with a unique mutation, which changes the sequence to a restriction enzyme site.

This construct is our timepoint 2 recording plasmid which contains the target sequence and 2 repeating units of DNA.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 3
  
• 1000
  COMPATIBLE WITH RFC[1000]