Difference between revisions of "Part:BBa K1150033"

Latest revision as of 23:15, 4 October 2013

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

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 6
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 1422
Illegal BamHI site found at 1379
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE 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.

Fig. 1: GFP Reporter in HEK293 cells

To use the GFP in the uniCAS toolkit, team Freiburg showed repression of fluorescence by a dCas9-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.

Fig. 2: GFP Reporter repressed in HEK293T cells. upper row: without Cas9, lower row: with dCas9-KRAB binding to the promoter of the reporter plasmid

@@ Line 20: / Line 20: @@
 |[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.
+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 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.
+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 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.
+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.
+Last but not least the plasmid can also be used for ''GFP'' expression in prokaryotic cells.
 <span class='h3bb'>
@@ Line 36: / Line 36: @@
 ===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. [[File:Freiburg2013-PIG6000-pIG0016-emx1-18.9-well6.jpg|thumb|300px|GFP Reporter in HEK293 cells]]
+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. [[File:Freiburg2013-PIG6000-pIG0016-emx1-18.9-well6.jpg|thumb|300px|Fig. 1: GFP Reporter in HEK293 cells]]
-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! [[File:Freiburg-2013 Gfp-repression.JPG|thumb|300px|GFP Reporter in HEK293t cells. upper row:without Cas9, lower row: with Cas9-KRAB binding to the promoter of the reporterplasmid cotransfected]]
+To use the GFP in the uniCAS toolkit, team Freiburg showed repression of fluorescence by a dCas9-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. [[File:Freiburg-2013 Gfp-repression.JPG|thumb|600px|Fig. 2: GFP Reporter repressed in HEK293T cells. upper row: without Cas9, lower row: with dCas9-KRAB binding to the promoter of the reporter plasmid]]