Team:TU-Delft/part3

Content

Introduction
Parts
Results
Conclusions
References

Introduction

Yeast with olfactory receptor+reporter=Snifferomyces

By combining the olfactory receptor and the FUS1pr-EGFP reporter, a complete yeast olfactory system is obtained. If the corresponding ligand binds to the receptor the FAR1 promoter is turned on and the EGFP is expressed. This EGFP signal can be read out by a fluorescence meter. If the olfactory system will be implemented as a diagnostics tool in developing countries, the EGFP reporter should be changed by a visible reporter.

Growth arrest

Besides the induction of the FUS1 promoter the cells also go in growth arrest mediated by the FAR1 promoter. However it is undesirable that the cells stop growing once they respond to a ligand. Therefor it is needed to knock out the FAR1 promoter.

Increasing sensitivity

To optimize the signal transduction from the receptor to the downstream cascade, a mammalian alpha subunit can be introduced which has affinity with the RI7-receptor [1]. For this, making a knockout of the native GPA1 gene is needed in order to let the subunit work as substitute. In the results we describe how we generated a knockout in yeast. In future work the alpha subunit should be expressed and characterized.

Parts

The receptor constructs and the reporter constructs are combined to have one complete olfactory system. The following biobricks are created:

BBa_K775005
BBa_K775006
BBa_K775007
BBa_K775008

Results

Transformations

Yeast strains were successfully transformed with the GPR109A receptor and output BBa_K775005 and the R17-ODR10 receptor and output BBa_K775008.

Fluorometer experiment

Setup

With yeast strains transformed with the GPR109A receptor and output BBa_K775005 and the R17-ODR10 receptor and output BBa_K775008 a fluorometer experiment was performed. After addition of the ligands (+ control alpha pheromone) OD600 and fluorescence were measured in time.

Outcome

Until the Wiki-freeze no observation of response to both constructs has been observed. This experiment is to be optimized to be able to draw a definite conclusion.

Conclusions

Four olfactory system biobricks were added to the registry. Two of the biobricks were successfully cloned in yeast. To draw conclusive conclusions about the systems further experiments are needed.

References

[1] Jasmina Minic, Marie-annick Persuy, Elodie Godel, Josiane Aioun, Ian Connerton, Roland Salesse, Functional expression of olfactory receptors in yeast and development of a bioassay for odorant screening, FEBS Journal (2005)