Team:TU-Delft/part3

From 2012.igem.org

(Difference between revisions)
Line 60: Line 60:
Flow cytometry data of +NR1 cells induced with nicotinic acid or alpha pheromones is shown.
Flow cytometry data of +NR1 cells induced with nicotinic acid or alpha pheromones is shown.
</p>
</p>
-
<img src=”http://2012.igem.org/wiki/images/c/c4/Flow_Cytometry_Snifferomyces_NR1_graph.png” alt="FC results" width=”350”>
+
<img src=”http://2012.igem.org/wiki/images/c/c4/Flow_Cytometry_Snifferomyces_NR1_graph.png” width=”350”>
<p>
<p>
Here we see that addition of receptor ligand doesn’t induce a response in comparison with alpha induced cells. Also in terms of a cutoff comparison in terms of percentages this seems to be the case. Possible explanations are:
Here we see that addition of receptor ligand doesn’t induce a response in comparison with alpha induced cells. Also in terms of a cutoff comparison in terms of percentages this seems to be the case. Possible explanations are:

Revision as of 22:31, 26 October 2012

Menu

Receptor

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. Therefore it is needed to knock out the FAR1 promoter. Therefore we characterized the knockout of 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

Characterization Far1 Knockout

The FAR1 has been knocked out to prevent growth arrest once a ligand has been added. To test this we have measured the optical density of Wild Type and Knockout strains with and without alpha feromones.

The results seem to show that there is a reduction in growth in both strains after the addition of alpha-feromones, although the Δfar1-strain shows less reduction.
Another remarkable feature is that the Δfar1-strain generally appears to grow faster than the Wild-type strain. A possible explanation is that the gene for growth-arrest has been knocked out. The FAR1 is not only related to the mating response, but is also a control in the mitotic cycle of budding yeast. [2]

Transformations

Yeast strains were successfully transformed with the GPR109A receptor and output BBa_K775005, called NR1 (nicotinic acid reporter). The RI7-ODR10 receptor and output BBa_K775008. Olfr154 receptor and output BBa_K775010, called BR1 (banana reporter 1) and RI7-OR1G1 receptor and output BBa_K775011, called BR2 (banana reporter 2).


Niacin Snifferomyces


Setup

In order to test the yeast NR1 cells with nicotinic acid receptor and the receptor inducible GFP reporter we induced the cells with nicotinic acid or alpha pheromone and measured the cellular response through flow cytometry.
Outcome

Flow cytometry data of +NR1 cells induced with nicotinic acid or alpha pheromones is shown.

Here we see that addition of receptor ligand doesn’t induce a response in comparison with alpha induced cells. Also in terms of a cutoff comparison in terms of percentages this seems to be the case. Possible explanations are:

  • A too small subset of the yeast receptors is localized in the membrane.
  • The affinity of the receptor with the alpha subunit is too low.
  • Isoamyl acetate Snifferomyces

    Setup
    Outcome

    Conclusions

    Four olfactory system biobricks were added to the registry.


    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)
    [2] "A cell sizer network involving Cln3 and Far1 controls entrance into S phase in the mitotic cycle of budding yeast" Alberghina et al. November 1, 2004 // JCB vol. 167 no. 3 433-443