Part:BBa_K1150033
Constitutive GFP Reporter
| GFP Reporter | |
|---|---|
| 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 a constituive active GFP reporter plasmid with strong GFP expression of mamalian cells. Due to a fused NLS it is localized in the nucleus. The different parts of this device were amplified by PCR with primers with overlaps and ligated via a four-fragment Gibson assembly. At this step a NLS was introduced behind the acGFP to ensure fluorescence in the nucleus. For an easily exchange of the promoter a NheI cutting site was introduced in front of it and a SacII cutting site behind it. Behind the acGFP two cuttingsites, SalI and BamHI, were established to be able to insert a DNA cutting recognition site. This leads to the possibility of separating the NLS from the GFP leading to fluorescence within the cytosol of mammalian cells. After 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 the percentage of cells that have taken up plasmids. This GFP repoter plasmid can further be used to show a repressive effect on gene-expression. Team Freiburg used it to show [http://2013.igem.org/Team:Freiburg/Project/effector#repression decrease of fluorescence] intensity when the CMV promoter is targeted by dCas9 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 Fig. 1. Here 1.5 ng 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 0.75 µg DNA and imaged after 48 h. The repressive effect on the GFP reporter can be seen in Fig. 2.
| None |