Part:BBa_K1150033
Constitutive GFP Reporter
| dCas9 | |
|---|---|
| Function | GFP expression |
| Use in | Mammalian cells and procaryots |
| RFC standard | RFC 10 RFC25 compatible |
| Backbone | pSB1C3 |
| Submitted by | [http://2013.igem.org/Team:Freiburg Freiburg 2013] |
this is constituive active GFP reporter plasmid with intense GFP expression in the nucleus of mamalian cells. The different parts of this device were amplified by PCR with primer with overlaps and via a four-fragment Gibson assembly ligated together. By this overlap a NLS behind the acGFP was introduced to ensure florescence in the nucleus. To be able to easily exchange the promoter of the construct a NheI cutting site was introduced in front of the CMV promoter and a SacII cutting site behind the promoter. Behind the acGFP two cuttingsites, SalI and BamHI, were established to be able to insert a DNA cutting recognition site here. This leads to the possibility to separate the nls from the gfp leading to fluorescence of the cytosol of mammalian cells. Behind the bgh terminator HindIII and KpnI cuttingsites were introduced to substitute a multiple cloning site found in most commercial available plasmids.
Usage and Biology
This reporter plasmid can be used for several purposes. To assess transfection efficiency in mammalian cells the plasmid can simply be co-transfected. About 20h after transfection very bright green fluorescence shows what percentage of cells has taken up plasmids. This GFP repoter plasmid can further be used to show a repressive effect of gene-expression repression systems. Team Freiburg used it to show decrease of fluorescence intensity when the CMV promoter is targeted by Cas9 fused to KRAB which is responsible for repression. Last but not least the plasmid can also be used for gfp expression in prokaryotic cells.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 6
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1422
Illegal BamHI site found at 1379 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Functional Parameters
This device was tested several times by team Freiburg 2013. Serving as transfection control the fluorescence of the GFP reporter can be seen in Image xy. Here 1,5ng DNA of this plasmid were transfected into Hek 293t cells in a 6-well dish and imaged after 20h and 44h.
To use the GFP in the uniCAS toolkit, team Freiburg showed repression of fluorescence by a Cas9-KRAB fusion construct which targets the CMV promoter of the plasmid. 24-wells were transfected with xyng DNA and imaged after xyh. The repressive effect on the GFP reporter can be easily seen Image 2b!
| None |