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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). ''Figure 1: Origins of chromoprotein structures[3]'' == Rationale == Chromoproteins have been characterized, codon optimized and standardized in E. Coli before by the Uppsala 2011 team (http://parts.igem.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2011&group=Uppsala-Sweden). However, even though multiple different pigment coding genes can be found in the standard biological parts registry (partsregistry.org), 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 == The aim is to design and validate the applicability of a set of chromoproteins originating from corals in A. Niger.
1. Design a palette of chromoprotein transcriptional units for A. Niger
2. Transform A. Niger with constructs containing normal chromoprotein coding genes, as well as codon optimized chromoprotein coding genes.
3. Introduce mitochondrial retentional signal to the chromoprotein coding genes for targeted localization
4. Validate in co-transformations whether pigments could function as elective markers == Approach == Part I Design a palette of chromoprotein transcriptional units for Aspergillus niger

1. We obtained the XL1 Blue MRF’ E. coli strains from Braunschweig university, which contain plasmids with 4 chromoprotein genes (amilGFP, aeBlue, cjBlue and eforRed) respectively, and DH5α E. coli strain containing mRFP gene from Uppsala university. Further information on the aforementioned plasmids is supplied in appendix 1.

2. Standardize the background for expression of all chromoprotein constructs by transforming DH5α E. coli competent cell with all 5 chromoprotein encoding gene containing plasmids. Then colony-PCR to check succesful transformation.

3. Design primers for each chromoprotein gene and then do PCR to amplify coding sequences which are around 700bp. These primers contain restriction sites needed for ligation into our in-house E. coli to Aspergillus shuttle vector.

4. Digest both the PCR product and the in-house shuttle vector and ligate the compatible ends.

5. Transform DH5α E. coli competent cells with ligation product.

6. Inoculate the transformants (which have an ampicillin resistance gene) on the Amp+ plates to select for transformants.

7. Colony-PCR to check which transformants contain the in-house shuttle vector + chromoprotein encoding gene.

8. Check whether the sequence of the chromoprotein encoding genes has remained unchanged by sending it out for sequencing and comparing it to the original sequence from parts registry.

Part II Introduce retention signals to the chromoprotein encoding genes for targeted organelle localization

1. We designed and ordered a synthetic construct entailing a Aspergillus codon-optimized chromoprotein (eforRed) encoding gene with an optional N-terminal mitochondrial signal peptide sequence (g-blocks).

2. We obtained the full synthetic construct after Gibson assembly of the g-blocks.

3. The synthetic construct is placed in a pJET vector. Amplify the pJET plasmid and validate insertion construct by Colony-PCR.

4. Digest the pJET plasmids to obtain the codon-optimized chromoprotein encoding gene and the codon-optimized chromoprotein encoding gene plus N-terminal mitochondrial signal peptide sequence.

5. Digest the in-house shuttle vector and ligate it to the constructs from step 4.

6. Transform DH5α E. coli competent cells with the ligation product.

7. Inoculate the transformants (which have an ampicillin resistance gene) on the Amp+ plates to select for transformants.

8. Colony-PCR to check which transformants contain the in-house shuttle vector + chromoprotein encoding gene.

9. Check whether the sequence of the chromoprotein encoding genes has remained unchanged by sending it out for sequencing and comparing it to the original sequence from parts registry.

Part III Transformation chromoprotein constructs and expression in Aspergillus niger

1. Transform protoplasts from Aspergillus niger N593 with plasmids containing chromoprotein encoding genes, as well as codon-optimized chromoprotein encoding genes.
2. Since the in-house shuttle vectors contain the pyrA gene, allowing them to grow without uridin, we inoculate the transformed protplasts on the uridin- plates to select successful transformants via uridin dependence.
3. Culture the A. Niger transformants and check the colour of their mycelium.

4. Isolate fungus DNA from the mycelium and do PCR to validate insertion of the chromoprotein encoding gene.

Part IV Standardization of measurement of chromoprotein encoding gene induced colorization

1. Plate DH5α containing no plasmid, containing aeBlue and eforRed in shuttle vector, eforRed and aeBlue in pSCB3 plasmid.

2. Create ImageJ macro to standardize measurement of colorization. Useful for when the color is non-visible to the naked eye.

== Appendix == 1. information of the plasmids with chromoprotein genes

pSB1C3-J23100-B0032-amilGFP CDS (green) chromoprotein gene BBa_K592010: http://parts.igem.org/Part:BBa_K592010

pSB1C3-J23100-B0032-eforRed CDS (green) chromoprotein gene BBa_K592012: http://parts.igem.org/Part:BBa_K592012

pSB1C3-J23100-B0032-aeBlue CDS (green) chromoprotein gene BBa_K864401: http://parts.igem.org/Part:BBa_K864401

pSB1C3-J23100-B0032-cjBlue CDS (green) chromoprotein gene BBa_K592011: http://parts.igem.org/Part:BBa_K592011

mRFP CDS (green) chromoprotein gene BBa_E1010: http://parts.igem.org/wiki/index.php/Part:BBa_E1010

Within the constructs, pSB1C3 is a high copy number plasmid carrying chloramphenicol resistance (http://parts.igem.org/wiki/index.php/Part:pSB1C3); J23100 is a constitutive promoter family member. (http://parts.igem.org/Part:BBa_J23100);B0032 is the ribosomal binding site (RBS) (http://parts.igem.org/Part:BBa_B0032).

== 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.