Difference between revisions of "Part:BBa K1150033"

Revision as of 09:19, 24 September 2013

Constitutive GFP Reporter

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

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]

@@ Line 2: / Line 2: @@
 <partinfo>BBa_K1150033 short</partinfo>
-this is constituive active GFP reporter plasmid with intense GFP expression im mamalian cells.
+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.
--- All templates were received from AG Weber, BIOSS. 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.
+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.
---
 <span class='h3bb'>Sequence and Features</span>
 <partinfo>BBa_K1150033 SequenceAndFeatures</partinfo>
-<!-- Uncomment this to enable Functional Parameter display
 ===Functional Parameters===
 <partinfo>BBa_K1150033 parameters</partinfo>
 <!-- -->