Part:BBa_K2371003

C-T7

dCas9 is a catalytically dead Cas9 protein that can combine with target sequence with the help of guide RNA(sgRNA).T7 RNA polymerase is a widely used polymerase from the T7 bacteriophage. BGIC-Union split the T7 polymerase into two separate parts(NT7:BBa_2371002 and CT7:BBa_2371003) and connect each of them to dCas9 protein via a linker.{N-T7-dCas9(BBa_K2371000) and C-T7-dCas9(BBa_K2371001)}

Figure 2. The schematic illustration of paired dCas9 system(1). dCas9 is connected to one of the piece of split T7 RNA polymerase(NT7 and CT7). They will combine with sgRNA beforehand and constitute the split T7-dCas9-sgRNA complex.

Each complex by itself is inactive, but when the two complex attached to particular sites we choose for identification of special sequences, they will reassemble to form a completed active T7 polymerase and start transcription in the presence of T7 promotor in cell free environment. Thus, this paired report system can convert the signal of specific cancerous gene into various report signals in cell free system. (i.e. GFP, LacZ, RFP)

Figure 3. The schematic illustration of paired dCas9 system(2). With the presence of target DNA, each complex will bind with pre-designed sgRNA-binding site on the sequence. When the split T7 polymerase approach each other close enough, they will become active and start transcription by adding report gene with T7 promotor in cell free system. After transcription, the mRNA will be translated into report protein like GFP and RFP which generate signal output.

We provide the two individual split T7 RNA polymerase N-T7 and C-T7 each connected with a linker for other team to leverage them through other methods.

We conducted both PCR(VF + VR) and enzyme(XbaI and SpeI) check.

Figure 4. PCR check of part plasmid N-T7 and C-T7 using VF and VR as primer. The expected PCR result of C-T7 should be around 1300bp. The expected PCR result of N-T7 should be around 2000bp.

Figure 5. Enzyme check of part N-T7 and C-T7. XbaI and SpeI were used. The expected length for C-T7 should be about 1000bp. The expected length for N-T7 should be about 1700bp.

Sequence and Features

Assembly Compatibility:

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

  Illegal BamHI site found at 973
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]