Difference between revisions of "Part:BBa K2996706"
 
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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. 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.   
 
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. 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.   
  
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<center><b>Figure 1.</b> <i>Gene circuit </i> </center>
 
<center><b>Figure 1.</b> <i>Gene circuit </i> </center>
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===Results===
 
===Results===
 
<p>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.</p>  
 
<p>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.</p>  
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<center><b>Figure 3.</b> <i> Fluorescence intensity of three kinds transformants before and after tetracycline induction</i> </center>
 
<center><b>Figure 3.</b> <i> Fluorescence intensity of three kinds transformants before and after tetracycline induction</i> </center>
  

Latest revision as of 00:01, 22 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. 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.


Figure 1. Gene circuit


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 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]