Difference between revisions of "Part:BBa K3017065"
Line 5: Line 5:
 
<P>This construct is to test if sgRNA works in vivo to form a complex with dCas9, and suppress fluorescent protein levels. It also aims to provide a baseline of fluorescence and suppressed fluorescence level.</P>
 
<P>This construct is to test if sgRNA works in vivo to form a complex with dCas9, and suppress fluorescent protein levels. It also aims to provide a baseline of fluorescence and suppressed fluorescence level.</P>
  
−
<H2>Design</H2>
+
 
 
<P>This construct consists of an sgRNA for GFP expressed under a pBAD promoter and constitutively expressed GFP and a constitutively expressed dCas9 expression construct (Bba_K3017069).</P>
 
<P>This construct consists of an sgRNA for GFP expressed under a pBAD promoter and constitutively expressed GFP and a constitutively expressed dCas9 expression construct (Bba_K3017069).</P>
 
<P>By using an inducible promoter, we can compare the GFP level before and after turning on the arabinose-inducible pBAD promoter, thus evaluating the suppression effect caused by CRISPRi (brought about by the dCas9-sgRNA complex). The unsuppressed GFP level (before arabinose induction) also gives us a figure of how the GFP expression level would be affected when it is co-expressed with a dCas9 protein.</P>
 
<P>By using an inducible promoter, we can compare the GFP level before and after turning on the arabinose-inducible pBAD promoter, thus evaluating the suppression effect caused by CRISPRi (brought about by the dCas9-sgRNA complex). The unsuppressed GFP level (before arabinose induction) also gives us a figure of how the GFP expression level would be affected when it is co-expressed with a dCas9 protein.</P>

Revision as of 00:24, 21 October 2019


Construct for testing CRISPRi sgRNA suppression on fluorescence protein production

This construct is to test if sgRNA works in vivo to form a complex with dCas9, and suppress fluorescent protein levels. It also aims to provide a baseline of fluorescence and suppressed fluorescence level.


This construct consists of an sgRNA for GFP expressed under a pBAD promoter and constitutively expressed GFP and a constitutively expressed dCas9 expression construct (Bba_K3017069).

By using an inducible promoter, we can compare the GFP level before and after turning on the arabinose-inducible pBAD promoter, thus evaluating the suppression effect caused by CRISPRi (brought about by the dCas9-sgRNA complex). The unsuppressed GFP level (before arabinose induction) also gives us a figure of how the GFP expression level would be affected when it is co-expressed with a dCas9 protein.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 3147
    Illegal PstI site found at 4569
    Illegal PstI site found at 4773
    Illegal PstI site found at 4803
    Illegal PstI site found at 6015
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
    Illegal NheI site found at 2131
    Illegal NheI site found at 2303
    Illegal NheI site found at 2326
    Illegal PstI site found at 3147
    Illegal PstI site found at 4569
    Illegal PstI site found at 4773
    Illegal PstI site found at 4803
    Illegal PstI site found at 6015
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 2608
    Illegal BamHI site found at 2070
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 3147
    Illegal PstI site found at 4569
    Illegal PstI site found at 4773
    Illegal PstI site found at 4803
    Illegal PstI site found at 6015
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 3147
    Illegal PstI site found at 4569
    Illegal PstI site found at 4773
    Illegal PstI site found at 4803
    Illegal PstI site found at 6015
    Illegal NgoMIV site found at 3435
    Illegal NgoMIV site found at 4539
    Illegal NgoMIV site found at 4612
    Illegal NgoMIV site found at 5097
    Illegal NgoMIV site found at 6006
    Illegal AgeI site found at 1905
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 705
    Illegal SapI site found at 1887