Difference between revisions of "Part:BBa K1119008"
Line 4: Line 4:
 
In characterization of Mitochondrial Leader Sequence ([[Part:BBa_K1119001|BBa_K1119001]]), negative control was made by GFP generator that does not contains the CDS of MLS ([[Part:BBa_K1119008|BBa_K1119008]]). The translation unit was driven by CMV promoter ([[Part:BBa_K1119006|BBa_K1119006]]) and terminated by hGH polyA signal ([[Part:BBa_K404108|BBa_K404108]]).  
 
In characterization of Mitochondrial Leader Sequence ([[Part:BBa_K1119001|BBa_K1119001]]), negative control was made by GFP generator that does not contains the CDS of MLS ([[Part:BBa_K1119008|BBa_K1119008]]). The translation unit was driven by CMV promoter ([[Part:BBa_K1119006|BBa_K1119006]]) and terminated by hGH polyA signal ([[Part:BBa_K404108|BBa_K404108]]).  
  
−
Since only short 5' untranscribed region ie. 6bp is included in the CMV promoter, future user may need to include spacer between CMV promoter and CDS to ensure successful translation.
+
 
  
 
===Result===
 
===Result===

Revision as of 12:24, 26 September 2013

CMV promoter - GFP - hGH polyA tail

In characterization of Mitochondrial Leader Sequence (BBa_K1119001), negative control was made by GFP generator that does not contains the CDS of MLS (BBa_K1119008). The translation unit was driven by CMV promoter (BBa_K1119006) and terminated by hGH polyA signal (BBa_K404108).


Result

In the characterization of CMV promoter (BBa_K1119006) , the coding sequence of CMV promoter was assembled with GFP reporter (BBa_K648013) and hGH polyA terminator (BBa_K404108) using Freiburg’s RFC25 format.

The pCMV-GFP was then transfected into HEK293FT cells and in vivo green fluorescence signal was observed under confocal microscope.

The positive control was pEGFP-N1 (Clontech) that contains CMV promoter and EGFP reporter. A negative control was made by GFP generator that does not contain the CMV promoter.

The [http://2013.igem.org/Team:Hong_Kong_HKUST/characterization detailed protocol] of our characterization can be found in HKUST iGEM 2013 Wiki.

Figure 1. CMV promoter drives expression of GFP. HEK cells transfected with pCMV-GFP expressed GFP Signal. HEK cells transfected with the commercial pEGFP-N1 showed similar results, while the negative control GFP without any promoter was not expressed.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 614
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 628
    Illegal AgeI site found at 1369
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1772
    Illegal BsaI.rc site found at 1274