E.Cargo

Results / Future Directions

Due to the gradual evolution of this project and the time constraints of our summer, we were unable to secure experimental data on fluorescence after assembling most of our constructs in the lab. That being said, we did plan a series of experiments to determine the efficiency of our Tat-TF delivery method.

Our first step is to confirm our reporter constructs work as expected. We created a construct that allowed cells to constitutively produce one of our transcription factors, LacI (BBa_K324003). When we transform our LacI reporter cell line with this construct, we expect LacI to be produced on site and endogenously repress cyan FP production. Quantitative analysis using a fluorometric plate reader in our research facility would measure any changes in fluorescence relative to background levels. This would be repeated with AraC to verify function of our inducible reporter.

The next stage of our project is to test Tat-TF's ability to translocate across the cell membrane. We plan to regrow the cell lines containing our reporter constructs, apply purified Tat-TF, and measure any changes fluorescence. We expect to see a decrease in cyan fluorescence in our repressible LacI reporter, and an increase in cherry fluorescence in our inducible AraC reporter. This would indicate that our cells are responding to the remote delivery of a transcription factor.

Finally, we will prove that the Tat domain is responsible for membrane translocation by lysing some of our cells that are constitutively producing LacI and AraC. We will apply the lysate, containing our transcription factor, to our reporter cell lines and look for changes to fluorescence. Lack of induction/repression would show that ordinary transcription factors lack the ability to freely enter the cell nucleus. This would suggest that our Tat domain functions as expected.