Part:BBa_K364333

TRE-Gal4-EcR-PolyA in PSB1A3 (expression vector)

Expression vector from standard BioBrick parts, expressing Gal4 - Ecdysone receptor LBD composite part.

The Gal4-EcR Chimeric nuclear receptor is an artificial eukaryotic TF made of Gal4 DBD (DNA Binding Domain) and D. Melanogaster nuclear hormone receptor LBD (Ligand Binding Domain) which can be expressed by this expression vector.

The minimal CMV promoter ensures a continuous expression of the chimeric construct, but furthermore it can be expressed in an inducible form using the Tetracyclin-controlled transcriptional activation system.

EcR LBD

The ecdysone receptor is a nuclear receptor found in arthropods, where it controls development and contributes to other processes such as reproduction. The receptor is a non-covalent heterodimer of two proteins, the EcR protein and ultraspiracle protein (USP). It binds to and is activated by ecdysteroids. Pulses of 20-hydroxyecdysone occur during insect development, whereupon this hormone binds to the ecdysone receptor, a ligand-activated transcription factor found in the nuclei of insect cells. This in turn leads to the activation of many other genes, which ultimately causes physiological changes that result in ecdysis (moulting).

Gal4 DBD

This protein is a positive regulator for the gene expression of the galactose-induced genes such as GAL1, GAL2, GAL7, GAL10, and MEL1 which encode for the enzymes used to convert galactose to glucose. This protein contains a fungal Zn(2)-Cys(6) binuclear cluster domain.

TRE-CMV

The Tetracycline Response Element (TRE) is recognized and bound by the Tetracycline repressor (TetR) protein. The TRE consists of 7 repeats separated by spacer sequences and placed upstream of CMV minimal promoter that has basal expession in the abscence of bound TetR. Tetracycline derivatives (e.g. doxycycline) bind TetR and render it incapable of binding to TRE, thereby forcing the expression of target genes.

PolyA

The PolyA tail of mRNA has multiple adenilates which is important for the nuclear export, translation and stability of mRNA in eukaryotes.

This composite artificial transcription factor will activate any reporter or any gene in general that has a UAS (Upper Activating Sequence) 3' of it's promoter. The usual binding sites of reporters, contain multiple UAS elements. In order to have a POPS output, the LBD has to recruit activators in the cell. This can be initiated by ligand binding or by recruiting a protein that has a fused strong activator like the VP activator.

With this system NHR (Nuclear Hormone Receptor) ligands or NHR interacting partners can be screened.

The NHR: cofactor-VP interaction should be also broken by a potential ligand binding, this is why this setup is also suitable for ligand identification. The benefit of the cofactor-VP interaction test is that the dynamic range of the assay is much higher than the dynamic range of the normal Gal4-NHR ligand activation assay.

More info about this project on the wiki pages of Team Debrecen-Hungary 2010. [http://2010.igem.org/Team:Debrecen-Hungary]

Picture of gel electrophoresis: TRE-Gal4-EcR-PolyA in pSB1A3 results a new expression vector from standard Biobrick parts. The TRE-Gal4-EcR LBD-PolyA insert is 1589 bp long, without polyA which was inserted in the last step is 191 bp shorter.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1461
  Illegal XhoI site found at 547
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 466