Difference between revisions of "Part:BBa K838001"
 
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The part consists of a TRP repressor and DsRed. The LovTAP-Vp16 protein attaches to the this readout and promotes transcription of the DsRed mRNA.
 
The part consists of a TRP repressor and DsRed. The LovTAP-Vp16 protein attaches to the this readout and promotes transcription of the DsRed mRNA.
  
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<!-- Add more about the biology of this part here
 
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===Usage and Biology===
 
  
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===Usage and Biology===
  
  
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Any promoters already present must be removed since we want only want expression drive by LovTAP-VP16 and not constitutive expression. The part must have a polyA tail at the end as well.  
 
Any promoters already present must be removed since we want only want expression drive by LovTAP-VP16 and not constitutive expression. The part must have a polyA tail at the end as well.  
  
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[[File:Team_EPF_Lausanne_simpleswitch.png|500px]]
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[[Image:500px-Team_EPF_Lausanne_simpleswitch.png]]
  
  
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Co-transfect the combination of LovTAP-Vp16 and readout in mammalian cells!
 
Co-transfect the combination of LovTAP-Vp16 and readout in mammalian cells!
  
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'''3) Illumination'''
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Continuous illumination with blue light (468 nm) is recommended for the LovTAP protein.
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To build a set up like the one we used, take a look at our [http://2012.igem.org/Team:EPF-Lausanne/Bioreactor Bioreactor assembly page], where we explain what components you need, how to assemble them and how to program your illumination device.
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'''4) Test the expression'''
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Since this readout system is built to express dsRed, a fluorescent protein, difference in fluorescence between cells in light or dark state can be measured using flow cytometry.
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This system still needs experimental optimisation, figure out how long the cells need to be illuminated, if they need a resting time in the dark, when exactly the dsRed is most expressed etc.
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Look at the Experience page and our [http://2012.igem.org/Team:EPF-Lausanne wiki] for more infos!
  
  
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K838001 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K838001 SequenceAndFeatures</partinfo>
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Latest revision as of 06:55, 6 October 2012

LovTAP readout This part needs to be used with part BBa_K838000, which is LovTAP-VP16.

The part consists of a TRP repressor and DsRed. The LovTAP-Vp16 protein attaches to the this readout and promotes transcription of the DsRed mRNA.


Usage and Biology

1) Cloning:

First clone the part into a mammalian expression vector such as [http://products.invitrogen.com/ivgn/product/V79020?ICID=search-product pcDNA3.1(+)] or [http://products.invitrogen.com/ivgn/product/V04450 pCEP4]. These are the two main expression vectors we used.

Any promoters already present must be removed since we want only want expression drive by LovTAP-VP16 and not constitutive expression. The part must have a polyA tail at the end as well.

500px-Team EPF Lausanne simpleswitch.png


2) Transfection Co-transfect the combination of LovTAP-Vp16 and readout in mammalian cells!

3) Illumination Continuous illumination with blue light (468 nm) is recommended for the LovTAP protein. To build a set up like the one we used, take a look at our [http://2012.igem.org/Team:EPF-Lausanne/Bioreactor Bioreactor assembly page], where we explain what components you need, how to assemble them and how to program your illumination device.


4) Test the expression Since this readout system is built to express dsRed, a fluorescent protein, difference in fluorescence between cells in light or dark state can be measured using flow cytometry. This system still needs experimental optimisation, figure out how long the cells need to be illuminated, if they need a resting time in the dark, when exactly the dsRed is most expressed etc. Look at the Experience page and our [http://2012.igem.org/Team:EPF-Lausanne wiki] for more infos!


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 424
    Illegal SpeI site found at 295
    Illegal SpeI site found at 303
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 424
    Illegal SpeI site found at 295
    Illegal SpeI site found at 303
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 424
    Illegal BglII site found at 381
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 424
    Illegal SpeI site found at 295
    Illegal SpeI site found at 303
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 424
    Illegal SpeI site found at 295
    Illegal SpeI site found at 303
    Illegal NgoMIV site found at 7
  • 1000
    COMPATIBLE WITH RFC[1000]