Difference between revisions of "Part:BBa K838000"
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When light hits the LovTAP protein, a conformational change happens and it can bind to the Trp binding site. With our activator system, transcription of the target gene is activated when cells are exposed to blue light. | When light hits the LovTAP protein, a conformational change happens and it can bind to the Trp binding site. With our activator system, transcription of the target gene is activated when cells are exposed to blue light. | ||
| − | === How do I | + | === How do I implement this switch in my mammalian cells? === |
| − | It's quite easy, all one needs to use this system is this part BBa K838000, [https://parts.igem.org/Part:BBa_K838001 BBa K838001] and some standard mammalian expression vector | + | It's quite easy, all one needs to use this system is this part BBa K838000, [https://parts.igem.org/Part:BBa_K838001 BBa K838001] and some standard mammalian expression vector to clone these two parts into. |
'''1) Cloning:''' | '''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. | 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. | ||
| + | '''Note:''' For the readout constructs it is highly recommended to cut out the CMV or other constitutive promoter from the mammalian vector. | ||
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. | ||
LovTAP Mammalian + dsRed readout [https://parts.igem.org/Part:BBa_K838001 BBa K838001] | LovTAP Mammalian + dsRed readout [https://parts.igem.org/Part:BBa_K838001 BBa K838001] | ||
| + | |||
| + | |||
| + | |||
| + | '''2) Transfection''' | ||
| + | Co-transfect the combination of LovTAP-Vp16 and readout in mammalian cells. | ||
[[Image:500px-Team_EPF_Lausanne_simpleswitch.png]] | [[Image:500px-Team_EPF_Lausanne_simpleswitch.png]] | ||
| + | '''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''' |
| − | + | The best way to check the presence of LovTAP-VP16 in the cells is to perform a Western Blot using an anti-VP16 antibody and a GAL4-VP16 positive control. | |
| + | It is also possible to exploit the autofluorescence of the Lov2 domain, a feature permitting to detect an emission wave length of 500nm when the domain is excited at 380nm or 490nm. Flow cytometry is a good technique for this. | ||
| + | Microscopy can be used to see if the protein is imported in the nucleus. | ||
| + | Look at the Experience page and our [http://2012.igem.org/Team:EPF-Lausanne wiki] for more infos! | ||
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Latest revision as of 06:45, 6 October 2012
Mammalian LovTAP-VP16
LovTAP-VP16 is a light-sensitive protein that binds to DNA. The protein is a fusion between a Lov light activated domain, a TRP repressor, an NLS/ linker domain and a VP16 activator domain. As it contains a VP16 activational domain, it's able to act as a transcriptional activator when it is bound to a TRP promoter site.
The lov domain clashes with the TRP repressor domain and prevents binding before the protein is exposed to blue light. After light activation the Lov domain goes through a comformational change and the TRP repressor domain of the protein can bind DNA.
Source
This part is an improvement of part BBa_K191006 designed by the 2009 EPF-Lausanne team (LovTAP, a light-sensitive repressor expressed in bacteria).
Usage and Biology
When light hits the LovTAP protein, a conformational change happens and it can bind to the Trp binding site. With our activator system, transcription of the target gene is activated when cells are exposed to blue light.
How do I implement this switch in my mammalian cells?
It's quite easy, all one needs to use this system is this part BBa K838000, BBa K838001 and some standard mammalian expression vector to clone these two parts into.
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. Note: For the readout constructs it is highly recommended to cut out the CMV or other constitutive promoter from the mammalian vector.
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.
LovTAP Mammalian + dsRed readout BBa K838001
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
The best way to check the presence of LovTAP-VP16 in the cells is to perform a Western Blot using an anti-VP16 antibody and a GAL4-VP16 positive control.
It is also possible to exploit the autofluorescence of the Lov2 domain, a feature permitting to detect an emission wave length of 500nm when the domain is excited at 380nm or 490nm. Flow cytometry is a good technique for this.
Microscopy can be used to see if the protein is imported in the nucleus.
Look at the Experience page and our [http://2012.igem.org/Team:EPF-Lausanne wiki] for more infos!
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 274
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 772
Illegal XhoI site found at 817 - 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 274
- 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 274
- 1000COMPATIBLE WITH RFC[1000]