Part:BBa_K1456025

tetR-ODD-VP16

ODD (oxygen dependent degradation) domain, which is present in the HIF-1α (hypoxia inducible factor) protein that it activates in lower oxygen levels and breaks down in intermediate oxygen levels, is a protein domain that plays a key regulatory role in the transcription of the HIF-1α factor. In intermediate oxygen levels, the ODD domain of the HIF-1α protein is hydroxilized by the hydroxilase enzyme and the hydroxilized HIF-1α enzyme breaks down through ubiquitin attachment. Thus, the ODD causes the regulation of a transcription factor, which is active in hypoxic conditions and inactive in normoxic conditions. The Tet-Off is a strong system composed of two strong plasmids. Of the two plasmids that form this system, the TetR-VP16 fusion protein produced by the first plasmid acts as a transcriptionary factor regulating the Tet operator sequence of the second plasmid(pTRE). (TetR: DNA Binding Domain, DBD; VP16: Transactivating domain, TAD).
The Tet-Off system can be inhibited using tetracyclane.

In this part, through placing the ODD region of the HIF-1α in between the synthetic TetR - VP16 transcription factors (which are not present in mammallian cells and have been used in molecular biology experiments for a long time), the transcription factor was designed to gain sensitivity to oxygen.

Modeling

The main goal of this part, being sensitive of TetR – VP16 system to hypoxia. In order to achieve this, ODD added between TetR and VP16 components.In this way, TetR-ODD-VP16 complex can only initiate the transcription of only weak promoter in hypoxic conditions because of the degradation of the complex by proteasome in oxygen presence. By using this system, we created a novel true/ false mechanism which triggers protein synthesis dependent on oxygen by modifying that has already present pTRE-Tet Off system.
In order to realize how our system will work. We try to calculate how the protein synthesis will effect under different concentration of O2 at the TetR - ODD - VP16 system.
Using the SimBiology toolbox for Matlab we created a framework of the pTRE-Tet Off System (Figure 2).

The symbol decleration is:

X1: pCMV
Y: ODD (dependent on TetR – VP16)
X2 : [TetRE -PminiCMV] Complex
X2Y: ODD[TetRE -PminiCMV]
X3 : Luciferase
Z : PHD (prolyl hydroxylase)
YZ: Hyroxylated ODD

After creating the basic framework for the model we needed to create mathematical equations for each reaction with appropriate rate constants. These equations and the corresponding values are shown below in Tables 3 and 4

What did it show?

After modified the TetR-VP16 system by adding ODD, we try to realize how our designed sytem will work under hypoxic and normoxic conditions. In normoxic condition, PDH is activated and hydroxylated the TetR-ODD-VP16 system, luciferase expression decreases.
In our model approach, we showed that luciferase expression increased %10 after exposed to 1nM O2 compared to 100 nM O2 concentration.

Modelling Results

Experimental Results:

We reached expected result in wet lab for HEK293 cell line. However, after we used HEPG2 cell line for this system, we had more reliable result about hydroxylation of ODD. The reason of this can be about life time of cells under hypoxia, because it will affect amount of PHD that enter nucleus during normoxia /hypoxia.

When the Tet Off-ODD and pTRE-Luc vectors were cotranfected to Hep G2 cells in hypoxic medium, there was a 4 times increase in Luciferase concentration in respect to normoxic medium.

In the light of these results, it is possible to say that the Tet-ODD-VP16 system was successfully synthesized and a novel hypoxia inducible system was introduced for future use in studies indulged into examining hypoxic conditions.

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal XbaI site found at 4
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
INCOMPATIBLE WITH RFC[23]
Illegal XbaI site found at 4
25
INCOMPATIBLE WITH RFC[25]
Illegal XbaI site found at 4
1000
COMPATIBLE WITH RFC[1000]