Team:Aberdeen Scotland/General Overview

Project overview

For iGEM2010, our team decided that the biology project would aim to produce a genetic toggle switch, which operated by controlling gene expression at the translational level. To implement the switch, two gene expression constructs were established, each consisting of an RNA-binding protein fused to either Green (GFP) or Cyan (CFP) fluorescent protein. When co-expressed in yeast, these translational fusions would be mutually inhibitory at the translational level, thereby forming a biological, ‘Toggle Switch’ system as shown by diagram 1.

‘Repeat the diagram here of the two constructs, showing RNA-level inhibition’

We have called this the ‘AyeSwitch’ and it is regulated by controlling the two constructs via different inducible yeast promoters, either GAL1 or CUP1 via the presence or absence of galactose and Cu2+ ions respectively. (A detailed description of how the AyeSwitch works is provided in our General Overview.)

Some of the important questions regarding our AyeSwitch were:

(i) Would this switch exhibit ‘bi-stability’ (i.e. could it switch from one state to another and back depending on the inducer present) (ii) Would this bi-stability switching be dose dependent (iii) What would happens if both inducers were present in equal concentration and length of time (iv) Could the bi-stability sensitivity be ‘fine-tuned’ using different mRNA stem loops and binding proteins

Since this translationally regulated toggle switch is a novel design, the project started with pilot experiments to determine the promoter responses to their respective inducing agents. This was successfully achieved using the genomically integrated constructs in which GFP gene was placed under the control of GAL1 or CUP1 promoter. The results are described in the ‘Promoter characterisation’ section.

Following this, we set out to test the two central constructs of our project (see Fig 1 above). As explained in the Results section, only one of these (GAL1p-[Npeptide-GFP]) was successfully expressed (see Switch characterisation). As a result we characterised the dose- and time-dependent induction of GAL1p-[Npeptide-GFP] using FACS analysis and fluorimetry.

In the remainder of the project, as described in the Switch troubleshooting section, we carried out a series of carefully designed cassette replacement experiments to identify the component in the CUP1p-[MS2-CFP] construct that was preventing CFP expression. This involved microscopy and fluorimetry (see Troubleshooting CUP1p-[MS2-CFP]). From these experiments, we concluded that the Bbox stem loop or fusion of MS2 coat binding protein to CFP may have prevented the expression of CFP.

Finally, we characterised a pre-existing bioBrick using one of our constructs, by replacing GFP in the functioning expression construct, GAL1p-[Npeptide-GFP], with mOrange (E2050) using homologous recombination and transformation into yeast. The results from this experiment showed that no mOrange fluorescence was observed from this modified construct. (Link to mOrange testing & Registry of Parts)