Part:BBa_K2996706
mRFP downstream of activation unit containing target
To facilitate measurements, we used a reporter plasmid pResponse-RFP, with the mRFP gene under the control of a weak promoter (BBa_J23117) that is preceded by a sequence rich in NGG PAM sequences on the NT strand and gRNA complements 20nt upstream of promotor J23117, so florescence intensity can be used to measure the degree of transcription activation.
Figure
We designed sgRNA that complements the NT strand from at least 20nt upstream from -55 point (With the transcription start denoted +1, RNAP complex covers from -55 to +20.)
When the dcas9-sgRNA complex binds to target sequence, RpoA will recruits RNA polymerase, promotor J23117 will be activated. Then, by measuring the biological activity of the downstream reporter gene, we will know the effectiveness of this transcription activation device under different conditions.
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 exponential 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. Florescence intensity
Figure 4. Bacteria culture after centrifuge
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 52
Illegal NheI site found at 75 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 39
Illegal AgeI site found at 661
Illegal AgeI site found at 773 - 1000COMPATIBLE WITH RFC[1000]
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