© 2018 UNSW Australia iGEM

Introduction

Förster Resonance Energy Transfer (FRET) is a method whereby the distance between two fluorophores can be calculated based on the amount of fluorescence signal emitted when excited. Every fluorophore has a range of wavelengths at which it can be excited, i.e. absorb electromagnetic energy, known as its absorption spectrum. After being excited, a fluorophore then emits energy at a range of wavelengths, known as its emission spectrum. The excitation wavelength is of higher energy (shorter wavelength) than the emission wavelength. The concept behind FRET is the excitation of one fluorophore which requires a high energy wavelength for excitation and another which requires a comparatively lower energy wavelength. The intensity of the second fluorophore's emission will depend on the distance that the emitted light from the first fluorophore has travelled (Figure 1). Therefore, intensity of the second fluorophore's emission represents fluorophore proximity. It is important to note that when selecting appropriate fluorophores, a large amount of overlap between the emission of the first fluorophore, in our case, mCerulean3, and the excitation of the second fluorophore, mVenus, is preferred.

As FRET has the ability to measure distances between fluorophores that are between 1 and 10 nm apart¹, by attaching mCerulean3 and mVenus to our Assemblase scaffold we hoped that FRET would occur between the scaffolded molecules. This would allow us to gain experimental data on the distance between the molecules that we scaffold, and hence more accurately calculate the diffusion patterns that would occur between enzymes that were scaffolded to our system.

Figure 1: Animation showing how fluorophores do not interact when too far apart, yet are able to transfer electromagnetic energy when brought close together.

Aim

Our aims were two-fold: 1. Firstly to perform FRET with unscaffolded mCerulean3 and mVenus in order to obtain their emission spectra. 2. To perform FRET with mCerulean3 and mVenus attached to our Assemblase scaffold to measure the distance between the molecules.

Due to time constraints, we were only able to meet our first aim of this experiment.

Methods

Solutions of mCerulean3 and mVenus were made up to 1 mg/mL and a serial dilution of each made with Phosphate Buffered Saline (PBS) from 1:1 through to 1:1000. The data presented by Markwardt et al.² and Jonáš et al.³ in the Figure 2 (below) was used as a starting point for determining the optimum excitation and emission wavelengths for the fluorescent proteins.

Figure 2: Excitation and emission spectra reported in the literature for mCerulean3 (A) and mVenus (B).

From this information from the literature, the following data (Table 1) was extrapolated as ideal values and ranges for mCerulean3 and mVenus excitation and emission.

Table 1: Ideal values and ranges of excitation and emission extracted from Figure 2.

These wavelengths were used to investigate the excitation and emission spectra of mCerulean3 and mVenus to determine the ideal wavelengths specific to our expressed proteins. Based on the resulting output graphs generated, curve smoothing was used before selecting an ideal excitation value that would maximise the excitation of mCerulean3 whilst minimising the excitation of mVenus.

Once this ideal value was obtained, mCerulean3 and mVenus were mixed in a 1:1 ratio, producing a solution containing each fluorescent protein at 0.5 mg/mL. Controls were also prepared using each fluorescent protein individually, made up to the same concentration with PBS. Using the ideal excitation value for cerulean determined previously, these wells were scanned to create the negative controls for future FRET experiments with scaffolded mCerulean3 and mVenus.