Part:BBa_K1023003

FRET based ATP biosensor cds, codon optimized for Aspergillus niger

The part is a codon optimized version of the ATP sensor dubbed as Ateam obtained from Imamura lab. The sensor consist of mseCFP (a variant of CFP), the ATP sensing domain is a subunit of the Bacillus subtilis FoF1-ATP synthase and mVenus (a YFP variant).

This sensor was codon optimized for Aspergillus niger and ordered as synthetic blocks and stitched together into pJET via Gibbson Assembly. This was verified by colony PCR. The sensor was then cloned into pSB1C3 and sequence verified before submission.

Usage and Biology

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.

Figure 1) Working of the ATP FRET sensor [1]

FRET based ATP bio-sensors, dubbed as Ateams, have been previously developed and implemented to quantify in vivo ATP levels in HeLa and yeast cells [1], [2] . The sensor consist of mseCFP (a variant of CFP), the ATP sensing domain is a subunit of the Bacillus subtilis FoF1-ATP synthase and mVenus (a YFP variant). The YFP/CFP emission ratio (527 nm/475 nm) gives an estimation of the ATP concentration. This sensor has been found to be ATP specific and pH independent, although it is temperature sensitive with a higher ATP binding capacity at lower temperatures. The Ateams have been requested from the Imamura lab in Japan for cloning and expression into A. niger and E. coli.

Reference:

1. Imamura, H., et al. (2009). "Visualization of ATP levels inside single living cells with fluorescence resonance energy transfer-based genetically encoded indicators." PNAS 106: 15651-15656.
2. Bermejo, C., et al. (2011). In vivo biochemistry: quantifying ion and metabolite levels in individual cells or cultures of yeast. Biochem. J 438 (1–10).

Sequence and Features BBa_K1023003 SequenceAndFeatures