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Introduction

Coral colorization occurs due to the presence of a range of fluorescent and non-fluorescent pigments. Fluorescent proteins (remotely homologous to the famous Green Fluorescent Protein superfamily from jellyfish[1], see Fig.1) are an interesting family of proteins. Coral fluorescent proteins are small, about 230 amino acid residues (~700bp) long, and acquired via evolution the ability to synthesize a chromophore from their own residues in a couple of autocatalytic reactions. Chromophore structure and kinetics are highly significant to the optics of colorizing proteins [2]. These proteins lead to myriad imaging techniques that capitalize their unique physical, biochemical and spectral properties. Non-fluorescent pigments are made up by chromoproteins, which absorb light effectively but hardly emit it. They have visible intrinsic colours which are visible to the naked eye. This feature, as well as their small gene size, make chromoproteins simple but favourable bio-reporters in molecular biology [1]. Most chromoproteins possess single absorption maxima around 560-590 nm. However, small shifts in the absorption maxima can already lead to compelling changes in the perceived colour; the pigment might even appear blue. Among GFP homologs, chromoproteins are quite unique in having the natural potential of far-red fluorescence (590-640 nm). However, far-red fluorescent proteins can now also be generated from chromoproteins via mutagenesis.[3] This property could lead to an interesting biotechnological application, as fluorescent near-infrared (650+ nm) reporters offer new possibilities for in vivo studies on biological functioning due to the high penetration of mammalian tissue by near-infrared light.

Mutagenesis or amino acid substitutions in chromoproteins can in addition lead to a huge increase (several hundred-fold) in their quantum yield and can colorization [4]. Chromophores in chromoproteins are usually characterized by a non-planar trans-conformation [5]. However, the chromoprotein colour differences are thought to occur due to interactions between the chromophore and environmental factors such as pH [6]. This finding is highlighted by results that show that a chromoprotein chromophore, though having the same amino acid sequence as a fluorescent protein, adopts a different conformation (deviation in chromophore ring planarity) under different environmental conditions [2, 5]. Chromoprotein structures are found in different lineages and have three independent origins, suggesting functional convergence [3] (Fig.1).

Fig.1 Origins of chromoprotein structures

Rationale

Chromoproteins have been characterized, codon optimized and standardized in E. Coli before by the Uppsala 2011 team. However, even though multiple different pigment coding genes can be found in the standard biological parts registry, none have been tested in filamentous fungus.

We now want to test the applicability of chromoproteins in Aspergillus Niger as part of a larger toolbox. This toolbox is aimed at making Aspergillus a more amenable host within synthetic biology and iGEM in particular. The chromoproteins can be used as reporter molecules, i.e. as elective markers in co-transformations. Besides that, they really make your organism look more cheery.

Aim

1.Design and validate the applicability of a set of chromoproteins originating from corals in Aspergillus Niger.
2.Introduce the mitochondrial retention signal to chromoprotein coding genes for targeted localization.
3.Standardize the measurement of chromoprotein encoding gene induced colorization.

Approach

The XL1 Blue MRF’ E. coli strains which contain plasmids with chromoprotein genes (amilGFP, aeBlue and eforRed) respectively and DH5α E. coli strain containing mRFP gene were obtained from Braunschweig UR and Uppsala UR. Then the palettes of those chromoprotein transcriptional units were designed for Aspergillus niger. Besides, mitochondrial retention signals to the chromoprotein encoding gene for targeted organelle localization was introduced via Gibson Assembling. Afterwards, each chromoprotein construct, including normal and codon optimized chromoprotein coding genes, was transformed with protoplasts and expressed in A. Niger N593. Additionally, the measurement of chromoprotein encoding gene induced colorization was standardized with ImageJ macro.

Results

The aeBlue, amilGFP, mRFP and eforRed chromoprotein genes were inserted into Funbrick succesfully. The new contructs were transformed with protoplasts and inoculated on CM- plates without uridine supplement. As shown in Fig.3, the mycilums didn't show corresponding colour as the chromoproteins in E.coli, see Fig.2.

However, PCR with the fungus DNA as templetes, and products dispalyed the expected bands which were around 700bp on gel electrophoresis, see Fig 4.

Via Gibson Assembling, the eforRed chromoprotein DNA was condon optimized and introduced with mitochondria retention signal peptide. The colony-PCR displayed the expected band on gel eletrophoresis, which is around 900bp, see Fig.5. Afterwards, the optimized construct will be validated in Aspergillus Niger.

Reference

1.Alieva, N.O., et al., Diversity and evolution of coral fluorescent proteins. Plos One, 2008. 3(7): p. e2680.
2.Chan, M.C., et al., Structural characterization of a blue chromoprotein and its yellow mutant from the sea anemone Cnidopus japonicus. Journal of Biological Chemistry, 2006. 281(49): p. 37813-37819.
3.Shagin, D.A., et al., GFP-like proteins as ubiquitous metazoan superfamily: evolution of functional features and structural complexity. Molecular biology and evolution, 2004. 21(5): p. 841-850.
4.Prescott, M., et al., The 2.2 Å crystal structure of a pocilloporin pigment reveals a nonplanar chromophore conformation. Structure, 2003. 11(3): p. 275-284.
5.Shkrob, M., et al., Far-red fluorescent proteins evolved from a blue chromoprotein from Actinia equina. Biochem. J, 2005. 392: p. 649-654.
6.Chalfie, M. and S.R. Kain, Methods of Biochemical Analysis, Green Fluorescent Protein: Properties, Applications and Protocols2005: Wiley-Liss.