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Introduction

Fluorescence resonance energy transfer (FRET) is a phenomenon widely exploited by bio-sensors to monitor concentrations and temporal fluctuations of metabolites and ions at cellular and sub-cellular level. FRET works by excitation of a fluorescent molecule (donor) by a light of particular wavelength, which consequently transfers this energy to an adjacent fluorescent molecule (acceptor) that in-turn emits light. This phenomenon is very sensitive to the distance between the donor and acceptor fluorophore groups. Thus, fusing fluorescent proteins with a sensing domain that undergoes big conformational changes upon binding of the sensory molecule confers the possibility for generating an assortment of custom-made genetically encoded biosensors. These are useful tools to non-invasively quantify metabolites in living cells.

ATP FRET sensor (Imamura et al. 2009)

FRET based sensors have been recently developed to quantify ATP levels in vivo in HeLa and yeast cells (1, 2). They consist of mseCFP, the ATP sensing domain and mVenus (YFP variant). The YFP/CFP emission ratio gives an estimation of the ATP concentration.

Aim

Construct a fluorescent protein based sensor that will measure ATP levels between compartments in growing Aspergillus niger during metabolic shifts.

Applications

An ATP bio-sensor can lead to visualization of the ATP levels in live Aspergillus cells. Inquiry into whether the single cell phenotype of Aspergillus niger still retains its metabolic activity can be carried out. It would be interesting to quantify the ATP levels in different organelles and investigate where and why such ATP levels exist. Also, the differences in cellular ATP under various growth environments can be studied. The differences in ATP levels for cells at the hyphal tip and those in the inner mycelium can be validated.

Approach

Introduce bio-sensor into A. niger directed to cytolpasm and mitochodria:

1.Codon optimized for A. niger
2.Not codon optimized

Express sensor in Escherichia coli and purify with the help of a Histidine tag for in-vitro characterization

Research Methods

The initial target was to introduce the following versions of the ATP sensor into A. niger – 1. ATP sensitive and cytoplasmic localization; 2. ATP sensitive and mitochondrial localization; 3. ATP in-sensitive and cytoplasmic localization; 4. Codon optimized, ATP sensitive and cytoplasmic localization; 5. Codon optimized, ATP sensitive and cytoplasmic localization.

Also, it was aimed to express the ATP sensor in Escherchia coli and purify with the help of a N-terminal Histidine tag for in-vitro characterization of the sensor. The in-vitro characterization of the purified sensor involves exciting the CFP with 435 nm light and measuring the YFP/CFP emission ratio (at 527/475 nm respectively) at different ATP concentrations. The characterization of the sensor was carried out at room temperature in a buffer mimicking the in-vivo conditions in A. niger.