Project Overview

Introduction

For this year’s iGEM competition the Aberdeen team has worked on developing a translationally controlled toggle switch embedded in yeast.^[1] Genetic toggle switches are a vital component for synthetic biology circuits , enabling functional control of biological functions. The majority of toggle switches used for iGEM are embedded in Escherichia coli and can only be controlled at the transcriptional level ^[2],[3]. Our main goal was to create and model a novel gene circuit, wherein yeast cells can be switched between mutually exclusive fluorescent proteins under exposure to environmental factors. This switching behaviour would be regulated at the translational level, an innovation over previous systems that only demonstrated transcriptional regulation ^[4],[5].The novel genetic toggle switch operated by controlling gene expression at the translational level consisted of two gene expression constructs expressing an RNA-binding protein fused to either Green (GFP) or Cyan (CFP) fluorescent protein in the presence of appropriate inducer. When co-expressed in yeast, these translational fusions would be mutually inhibitory at the translational level, thereby forming a biological, ‘Toggle Switch’ system.

The AyeSwitch

The toggle switch is shown by Fig 1 and was named the ‘AyeSwitch’. It is regulated by controlling the two constructs, GAL1p-[Npeptide-GFP] and CUP1p-[MS2-CFP], via inducible yeast promoters GAL1 or CUP1 in the presence or absence of galactose and Cu2+ ions respectively.

For example, in the presence of galactose only, GAL1 is induced and there is expression of N-peptide-GFP protein. The subsequent addition of Cu2+ then induces the transcription of mRNA coding for MS2 coat binding protein and CFP. In addition to this, the mRNA also codes for a Bbox stem loop sequence that can be bound by N-peptide.

Ideally, there is initial inhibition of MS2-CFP translation by Npeptide-GFP binding to the Bbox stem loop. Evolution of time corresponds to the ratio of MS2-CFP mRNA to N-peptide-GFP protein increasing allowing some MS2-CFP to be produced until CFP ‘switches ON’ as it gains dominance over GFP.

Additionally, N-peptide-GFP protein translation can also be inhibited by MS2-CFP via MS2 protein binding to the MS2 stem loops on the N-peptide-GFP mRNA. This may help the switching ON of CFP and also means GFP would face a similar situation if the inducer was changed from Cu2+ to galactose.

However, additional variables may come into play affecting the outcomes described above. It is likely that the concentration of each inducer present, the translational rate and binding efficiency of stem loop binding proteins to mRNA stem loop and degradation rate of proteins can also affect the outcome. Reversing the order of inducer present may also affect the outcome.

Experimental Characterisation of the AyeSwitch

The experimental work addressed these issues by initially characterising the promoters in terms of their dose response and time response using constructs GAL1-[GFP] and CUP1-[GFP]. These experiments were then extended to characterise GAL1p-[Npeptide-GFP] and CUP1p-[MS2-CFP] which discovered that CUP1p-[MS2-CFP] did not function as expected.

The experimental work diverged from this point to troubleshoot CUP1p-[MS2-CFP], investigating the translation inhibition of GAL1p-[Npeptide-GFP] by MS2 coat protein using construct MET17p - [MS2], Bio-brick construction and testing of Bio-brick E2050 mOrange.

Troubleshooting CUP1p-[MS2-CFP]

Troubleshooting of CUP1p-[MS2-CFP] was carried out through a series of gene cassette replacement experiments testing the promoter and CFP sequences for functionality. The conclusions to these experiments suggest that the Bbox Stem loop, usually located in the 3’untranslated region but is in the 5’ untranslated region of our construct may be preventing the expression of downstream proteins. It may also be that the fusion of MS2 to CFP results in inappropriate protein folding, inhibiting expression.

Verification of Translation Inhibition as a Regulatory Mechanism

It was shown that the translational inhibition of GAL1p-[Npeptide-GFP] by MS2 coat protein was possible, confirming that translational regulation is viable. Further work if time permitted would investigate if this inhibition could work in the context of a toggle switch.

Bio-brick construction and testing

In parallel, Bio-bricks were constructed and submitted to the Registry of parts whilst testing of the Bio-brick E2050 mOrange using fluorimetry and FACS analysis lead to the conclusion that the mOrange sequence did not function within our GAL1p-[Npeptide-GFP] construct that was shown to be able to express GFP appropriately.

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References

¹ Ernesto Andrianantoandro et al. Synthetic biology: new engineering rules for an emerging discipline Molecular Systems Biology 2:2006.0028

² Timothy S. Gardner et al. Construction of a genetic toggle switch in Escherichia coli Nature 403, 339-342 (20 January 2000)

³ Mariette R. Atkinson et al. Development of Genetic Circuitry Exhibiting Toggle Switch or Oscillatory Behavior in Escherichia coli Cell, Volume 113, Issue 5, 597-607, 30 May 2003

⁴ Adam Platt and Richard J Reece The yeast galactose genetic switch is mediated by the formation of a Gal4p–Gal80p–Gal3p complex The EMBO Journal (1998) 17, 4086 - 4091

⁵ D W Griggs and M Johnston Regulated expression of the GAL4 activator gene in yeast provides a sensitive genetic switch for glucose repression PNAS October 1, 1991 vol. 88 no. 19 8597-8601