Difference between revisions of "Part:BBa K1119008"

Revision as of 12:19, 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).

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

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