Difference between revisions of "Part:BBa K1150033"
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<partinfo>BBa_K1150033 short</partinfo>
 
<partinfo>BBa_K1150033 short</partinfo>
  
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{| style="color:black" cellpadding="6" cellspacing="1" border="2" align="right"
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! colspan="2" style="background:#FFBF00;"|GFP Reporter
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|-
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|'''Function'''
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|GFP expression
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|-
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|'''Use in'''
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|Mammalian cells and procaryots
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|-
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|'''RFC standard'''
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|[https://parts.igem.org/Help:Assembly_standard_10 RFC 10] RFC25 compatible
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|-
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|'''Backbone'''
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|pSB1C3<br>
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|-
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|'''Submitted by'''
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|[http://2013.igem.org/Team:Freiburg Freiburg 2013]
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|}
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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.
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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.
  
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===Usage and Biology===
 
===Usage and Biology===
  
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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.
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<span class='h3bb'>Sequence and Features</span>
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Last but not least the plasmid can also be used for ''GFP'' expression in prokaryotic cells.
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<span class='h3bb'>
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===Sequence and Features===
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<partinfo>BBa_K1150033 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K1150033 SequenceAndFeatures</partinfo>
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===Functional Parameters===
 
===Functional Parameters===
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<partinfo>BBa_K1150033 parameters</partinfo>
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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]]
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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]]

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