The assembled PA2652 construct (BBa_K515102) and non-codon optimised mcpS (in pRK415 backone vector), have been inserted and tested for functionality in E. coli DH5α obtained from New England Biolabs. We carried out several tests in an attempt to characterise the rewired chemotaxis towards L(-)malic acid. We separated testing of chemotaxis towards malate into behavioural, qualitative & quantitative analyses.

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We are currently testing the chemotactic response of our chassis to physiologically relevant malate concentration levels below 10^-6 M. To obtain these results we have reassessed the data collection method based on flow cytometry, because this should speed up the process of data collection and accuracy. To distinguish our bacteria from the background particles we have cotransformed our chassis with a low copy plasmid pTAC1 containing GFP, so our cells can be uniquely visualised using flow cytometer even if their size is similar to the size of backround particles. Hopefully this new strategy will produce results for presentation in Boston.

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ODD SYNTHESİS FROM HEP2G VİA PCR

ODD (Oxygen Dependent Degredation) domain of HIF-1α was synthesized through liver cDNA using PCR with Sall enzyme restriction cites placed at the starting and ending points of the domain.

The PCR product was purified using the Phenol Chloroform method. Following the isolation, the ODD and pTet-Off vector were cut using the Sall restriction enzyme and then ligated. Thus, the ODD insert was placed in between the tetR(DNA binding domain) and VP16(Transactivating domain) of the pTet-Off vector.

COLONY PCR

The DH-5α E.coli strains were transformated and, using CMV forward and SV40 polyA reverse primers, colony PCR was conducted and the vectors, in which the inserts were placed, were elected.

CUT-CHECK

Since the ODD insert’s contained the same restriction cite on both ends, the colonies that entered the sequence from the right end were cut-checked using EcoRI and BamHI restriction enzymes and the colony containing the desired vector was selected.

ODD Results

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 study, 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. The therapeutic genes in the pTRE genes can be synthesized as sensitive to the hypoxic conditions, controlled by the TetR-ODD-VP16 transcription factor.

Luciferase Assay

To control the functionality of the TetR-ODD-VP16 system, the HEK 293T and Hep G2 cell lines were cotransfected using the pTET Off-ODD and pTRE-Luc vectors.

100 µM of CoCl2 was added to the cell medium to establish 1% O2 in the medium. The cells were collected 9 hours later. Luminometric measurement under Thermo Varioscan for 613 nm was done and the data of the following graph were acquired:

The observation of the operational level of the TetR-ODD-VP16 did not yield fruitful results as the cells reached out of the dish when 100 µM of CoCl2 were added to HEK 293T medium.

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.

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