Part:BBa_K2996701

RpoA fused with dcas9 through FL4

This fusion protein is the transcription activator. Using overlap PCR, RpoA BBa_K2996701, is linked to dcas9 BBa_K2996706, through a modified flexible Linker 4, BBa_K2996700.

Based on this, we constructed plasmid pActivator without sgRNA and pActivator with sgRNA. When dcas9 binds to the target or lure sequence under the guidance of sgRNA, RNAP will activate adjacent promotor J23117. The transcription of downstream reporter gene is promoted.

Figure 1. pActivator diagram Figure 2. pResponse-RFP diagram

Results

We got three kinds of transformants, each harboring (a)pResponse-RFP, (b)pResponse-RFP plus pActivator without sgRNA, (c)pResponse-RFP plus pActivator with sgRNA. Overnight cultures of different colonies were transferred and grow into early logarithmic stage. Then, we added tetracycline to culture (b) and (c) to a final concentration of 100ug/ml, and incubated them overnight at 30℃. Samples were prepared according to protocol and loaded to 96-well plate. Below is the data acquired from microplate reader, with excitation wavelength at 584nm and emission wavelength at 607nm. Florescence/OD600 indicates the intensity of mRFP.

Figure 3. Fluorescence intensity of three kinds transformants before and after tetracycline induction

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 2161
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 4440
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 459
  
• 1000
  COMPATIBLE WITH RFC[1000]