Part:BBa_K626000

TRE-Bax-PolyA in PSB1A3 (expression vector)

Expression vector from standard BioBrick parts, expressing human pro-apoptotic protein BAX.

The human BAX is a pro-apoptotic protein which can be expressed by this expression vector.

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

hBAX

Bax (Bcl-2-associated X) protein is a pro-apoptotic member of the Bcl-2 protein family. It contains three BH (Bcl-2 homology) domains, can form homo- and heterodimers and plays essential role in the formation of MOMP (Mitochondrial outer membrane permeabilization) leading to apoptosis through cytochrome c release which is the most frequent type of PCD (Programmed cell death).

The action of Bax protein in programmed cell death.

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.

With this system PCD (Programmed Cell Death) can be induced in a regulated way.

Picture of gel electrophoresis: TRE-Bax-PolyA in pSB1A3 results in a new expression vector from standard Biobrick parts.

Picture of Western blot: Bax is highly expressed in the expression vector created from pSB1A3

Picture of DAPI staining: The nuclear fragmentation was clearly visible by Hoechst staining, which is a known marker of apoptosis.

Sequence and Features

Assembly Compatibility:

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

  Illegal BamHI site found at 778
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]