Difference between revisions of "Part:BBa K838000"
(→Source) |
(→Usage and Biology) |
||
| Line 19: | Line 19: | ||
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] | ||
| + | |||
| + | [[Image:500px-Team_EPF_Lausanne_simpleswitch.png]] | ||
| + | |||
| Line 29: | Line 31: | ||
<span class='h3bb'>Sequence and Features</span> | <span class='h3bb'>Sequence and Features</span> | ||
<partinfo>BBa_K838000 SequenceAndFeatures</partinfo> | <partinfo>BBa_K838000 SequenceAndFeatures</partinfo> | ||
| − | |||
| − | |||
===Functional Parameters=== | ===Functional Parameters=== | ||
<partinfo>BBa_K838000 parameters</partinfo> | <partinfo>BBa_K838000 parameters</partinfo> | ||
<!-- --> | <!-- --> | ||
Revision as of 07:42, 3 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 on Part:BBa_K838000 (LovTAP, a light-sensitive repressor expressed in bacteria).
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.
LovTAP Mammalian + dsRed readout BBa K838001
2) Transfection Co-transfect the combination of LovTAP-Vp16 and readout in mammalian cells!
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]