Difference between revisions of "Part:BBa K2996706"

Revision as of 16:11, 19 October 2019

mRFP downstream of activation unit containing target

To facilitate measurements, we used a reporter plasmid pResponse-RFP, with mRFP downstream of BBa_K2996704. Now, 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

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

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 52
Illegal NheI site found at 75
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 39
Illegal AgeI site found at 661
Illegal AgeI site found at 773
1000
COMPATIBLE WITH RFC[1000]

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K2996706 short</partinfo>
-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.
+To facilitate measurements, we used a reporter plasmid pResponse-RFP, with mRFP downstream of BBa_K2996704. Now, 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.