Part:BBa_K2065003

CT52-Caspase9

This BioBrick is a coding sequence for the CT52 protein, attached to the Caspase 9 protein with a GGS10 linker in between. Because CT52 has a binding interaction for the T14-3-3 scaffold protein under influence of the small molecule fusicoccin, it can be used for assembly of proteins on this scaffold protein.

Design of the construct This parts is constructed of three subparts; CT52, a GGS10 linker and Caspase 9. It has a molecular weight of 52 kDa. CT52 is the name for the isolated last 52 C-terminal amino acids of the plant plasma H+-ATPase PMA2 [1]. The N-terminus of CT52 is connected to a flexible GGS10 linker. This linker consists of a ten times repeating Glycine-Glycine-Serine amino acid sequence.

Usage Caspase 9 is an apoptosis inducer which can activates by dimerizing with itself. The ability of caspase 9 to cleave the Ac-LEHD-AFC substrate can be used to determine the caspase 9 activity. When two caspase-9 proteins dimerize, and cleave Ac-LEHD-AFC, this substrate splits into a non-fluorescent peptide and a fluorescent part. This fluorescence has an excitation of 400 nm and emission at 505 nm. Thus caspase 9 activity can be measured by means of fluorescence. [1] When caspase-9 and CT52 are linked, the binding affinity of CT52 and the T14-3-3 construct can be measured by means of fluorescence . Because caspase 9 is an apoptosis inducer so Caspase9-CT52 could also be used in a T14-3-3 based kill switch.

iGEM TU Eindhoven used this part to characterize a tetrameric variant of the T14-3-3 scaffold protein. Unfortunately constructing this protein did not succeed, so no experimental data was obtained from this protein.

Sequence The sequence of our Capase9-CT52 has been verified by StarSeq. It contains the prefix and suffix with the correct restriction sites (EcoRI, XbaI, SpeI and PstI). Caspase9-CT52 is 1386 bp long.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]