Team:Wageningen UR/Chromoproteins

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<h1>Chromoproteins</h1>
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<h2>Simple bioreporters in synthetic biology</h2>
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         <li class="fillfirst"><a href="https://2013.igem.org/Team:Wageningen_UR/Why_Aspergillus_nigem">Why Aspergillus nigem?</a></li>
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         <li class="firstbgsm fill"><a href="https://2013.igem.org/Team:Wageningen_UR/Why_Aspergillus_nigem">Why <i>Aspergillus nigem</i>?</a></li>
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        <li class="fill"><a href="https://2013.igem.org/Team:Wageningen_UR/Secondary_metabolites">Secondary metabolites</a></li>
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        <li class="fill"><a href="https://2013.igem.org/Team:Wageningen_UR/Secondary_metabolites">Secondary metabolites</a></li>
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        <li class="fill"><a href="https://2013.igem.org/Team:Wageningen_UR/Secondary_metabolites">Secondary metabolites</a></li>
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        <li class="fill"><a href="https://2013.igem.org/Team:Wageningen_UR/Lovastatin">Lovastatin</a></li>
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    <h1>Chromoproteins</h1>
 
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    <h2>Aspergillus Pigmenti</h2>
 
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== Introduction ==
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== Outline ==
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        <p>
 
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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.
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<p>We aim to establish <i>Aspergillus niger</i> as a promising host in synthetic biology and in iGEM in particular. Therefore we introduce a range of simple bio-reporters such as pH, ATP sensors and chromoproteins, to increase the usability of the <i>Aspergillus</i> toolbox. Here, we focus on chromoproteins which are pigments obtained from corals. Chromoproteins have been characterized, codon-optimized and standardized in <i>E. coli</i> before by the <a href="http://parts.igem.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2011&group=Uppsala-Sweden" target="_blank">Uppsala 2011 team</a>. However, even though multiple different pigment coding genes can be found in the <a href="http://partsregistry.org" target="_blank">standard biological parts registry</a>, none have been tested in filamentous fungi.</p>
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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).
 
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''Figure 1: Origins of chromoprotein structures[3]''
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== Introduction ==
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== Rationale ==
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<p>
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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[<a href="#ref1">1</a>], see Fig.1) are an interesting family of proteins. Coral fluorescent proteins are small, about 230 amino acid residues (~700bp) long. They 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 [<a href="#ref2">2</a>]. 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 intrinsic colour which is visible to the naked eye. This feature, as well as their relatively small gene size, make chromoproteins simple but favorable bio-reporters in molecular biology [<a href="#ref1">1</a>]. 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 color; 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). Furthermore, far-red fluorescent proteins can also be generated from chromoproteins via mutagenesis.[<a href="#ref3">3</a>] 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 their ability of penetrating mammalian tissue via near-infrared light.</p>
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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.
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<p>In addition, mutagenesis or amino acid substitutions in chromoproteins can lead to a huge increase (several hundred-fold) in their quantum yield and can influence colourization [<a href="#ref4">4</a>]. Chromophores in chromoproteins are usually characterized by a non-planar trans-conformation [<a href="#ref5">5</a>]. However, the differences in chromoprotein colour are thought to occur due to interactions between their chromophore and environmental factors such as pH [<a href="#ref6">6</a>]. 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 [<a href="#ref2">2</a>, <a href="#ref5">5</a>]. Chromoprotein structures are found in different lineages and have three independent origins, suggesting functional convergence [<a href="#ref3">3</a>] (Fig.1).
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</p>
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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.
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<img src="https://static.igem.org/mediawiki/2013/0/00/Chromo_origins_wiki.jpg" style="width:90%;height:80%;"/>
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<p class="caption"> Fig.1 Origins of chromoprotein structures </p>
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== Aim ==
== Aim ==
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The aim is to design and validate the applicability of a set of chromoproteins originating from corals in A. Niger.
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1. Design a palette of chromoprotein transcriptional units for A. Niger
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<br /><span class="ref"> 1.</span>Design and validate the applicability of a set of chromoproteins originating from corals in <i>Aspergillus niger</i>.  
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2. Transform A. Niger with constructs containing normal chromoprotein coding genes, as well as codon optimized chromoprotein coding genes.
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<br /><span class="ref">2.</span>Introduce the mitochondrial retention signal to chromoprotein coding genes for targeted localization.
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3. Introduce mitochondrial retentional signal to the chromoprotein coding genes for targeted localization
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<br /><span class="ref">3.</span>Standardize the measurement of chromoprotein encoding gene induced colorization.</p>
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4. Validate in co-transformations whether pigments could function as elective markers
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== Approach ==
== Approach ==
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<p>The XL1 Blue MRF’ <i>E. coli</i> strains which contain plasmids with chromoprotein encoding genes <a href="http://parts.igem.org/Part:BBa_K864401:Experience" target="_blank">aeBlue</a>, <a href="http://parts.igem.org/Part:BBa_K1073022" target="_blank">eforRed</a> and <a href="http://parts.igem.org/Part:BBa_K1073024" target="_blank">amilGFP</a>
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respectively and DH5α <i>E. coli</i> strain containing <ahref="http://parts.igem.org/Part:BBa_E1010" target="_blank">mRFP</a> were obtained from Braunschweig UR and Uppsala UR.
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Part I  Design a palette of chromoprotein transcriptional units for Aspergillus niger
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Then the palettes of those chromoprotein transcriptional units were tested for applicability in <i>Aspergillus niger</i>. Besides, we synthesized and then Gibson assembled a chromoprotein encoding gene <a href="http://parts.igem.org/Part:BBa_K1023005" target="_blank">(eforRed)</a>
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which was codon-optimized for <i>Aspergillus niger</i>. A mitochondrial retention signal was added to this codon-optimized chromoprotein encoding gene for targeted organelle localization. Afterwards protoplasts were transformed with all chromoprotein constructs, normal and codon optimized, in order to be expressed in <i>A. niger</i>  N593.</p>
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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.
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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.
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== Results ==
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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.
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<p>The aeBlue, amilGFP, mRFP and eforRed chromoprotein genes were succesfully inserted into an in-house vector. After their sequence was confirmed by single read sequencing, protoplasts were transformed with these chromoprotein encoding genes. Then they were inoculated on complete medium plates without uridine supplement. We thereby created selective pressure since the <i>A. niger</i> N593 strain owns a uridine autroxophy gene.
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4. Digest both the PCR product and the in-house shuttle vector and ligate the compatible ends.
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However, unlike the <i>E. coli</i> transformants (Fig.2) the <i>Aspergillus</i> transformant colonies (Fig.3) did not show colour.
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<img src="https://static.igem.org/mediawiki/2013/thumb/1/14/Fig2_wur_jing.PNG/800px-Fig2_wur_jing.PNG" style="width:100%;height:150%;"/>
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5. Transform DH5α E. coli competent cells with ligation product.
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<p>However, when performing PCR with the fungal genomic DNA as a template, the products appeared to be of the right size, giving the expected bands which were around 700bp after gel electrophoresis, see Fig 4.</p>
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6. Inoculate the transformants (which have an ampicillin resistance gene) on the Amp+ plates to select for transformants.
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<img src="https://static.igem.org/mediawiki/2013/thumb/e/ed/Fig_4.PNG/800px-Fig_4.PNG" style="width:100%;height:100%;"/>
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7. Colony-PCR to check which transformants contain the in-house shuttle vector + chromoprotein encoding gene.
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<p>The <a href="http://parts.igem.org/Part:BBa_K1023005" target="_blank">eforRed</a> chromoprotein encoding gene was codon-optimized for <i>A. niger</i> and synthetic construct blocks were ordered and fused via Gibson Assembly. This synthetic construct contains a removable mitochondrial targeting sequence. The colony-PCR of <i>E. coli</i> transformed with this codon-optimized eforRed with mitochondrial retention sequence displayed the expected bands after gel electrophoresis, which were around 750 bp, see Fig.5. Afterwards, <i>Aspergillus niger</i> was transformed with the codon-optimized construct with mitochondrial retention signal. Today (05-10-2013) we noticed that the mycelium of one of the aforementioned transformed <i>A. niger</i> showed red colour (Fig.6)!!! The day before the wiki-freeze, we got the result we were longing for for so long!
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<img src="https://static.igem.org/mediawiki/2013/archive/e/e1/20131004170251%21New_colour_wur_jing.PNG" style="width:100%;height:100%;"/>
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<br>
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We performed PCR on our construct in fungal DNA and on our construct in the biobrick backbone as a positive control this very evening before the wiki-freeze. It showed faint bands of the expected size (750 bp)(Fig.7)! <br>
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Lane 1 = marker, lane 2+3 = transformant fungal DNA, lane 5 = negative control, lane 7+8 = positive control (biobrick)
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<img src="https://static.igem.org/mediawiki/2013/7/7c/YEA_purple_%282%29.jpg" style="width:40%;height:40%;"/>
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Fig. 7 PCR of transformant <i>A. niger</i>
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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.
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Part II Introduce retention signals to the chromoprotein encoding genes for targeted organelle localization
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== Conclusions ==
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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).
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<p> The correct insertion of the non-coding optimized chromoprotein encoding genes in the fungus genomic DNA was confirmed by PCR. To further confirm whether the sequence of the chromoprotein encoding genes also remained unchanged, we sent the new constructs out for sequencing. Comparing the sequencing results to the original sequence from parts registry, for the aeBlue, mRFP and amilGFP chromoprotein gene, the identity was almost 100%. We thereby showed that the palettes of chromoprotein transcriptional units for <i>A. niger</i> were designed and transformed successfully. Additionally, we optimized the codon of eforRed chromoprotein encoding gene for <i>A. niger</i> and introduced mitochondrial retention signal for targeted localization. The transformation of <i>A. niger</i> with the optimized constructs was successfully validated via PCR and its mycelium showed the red colour as we expected. For the PCR however the bands for amplified transformant fungal DNA were not really intense compared to the bands for the amplified positive controls. The reason for this could be that our biobricking primers only partly annealed to our codon-optimized construct with signal sequence in the fungal DNA, while they fully anneal to our construct in biobrick vector. </p>
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2. We obtained the full synthetic construct after Gibson assembly of the g-blocks.  
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<p>To conclude, the codon optimized <a href="http://parts.igem.org/Part:BBa_K1023005" target="_blank">eforRed</a> chromoprotein could be introduced into <i>A. niger</i> and applied as a simple selective marker or bioreporter. For the others whose mycelium didn’t show colour, we assume that it is either related to transcription or translation of the chromoprotein encoding gene in <i>A. niger</i>. Future analysis, such as RT-PCR is suggested to check if the mRNA is transcribed properly. A good alternative would be to add a His tag, purify the chromoprotein and thereby determine translation.</p>
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3. The synthetic construct is placed in a pJET vector. Amplify the pJET plasmid and validate insertion construct by Colony-PCR.
 
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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.
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5. Digest the in-house shuttle vector and ligate it to the constructs from step 4.
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6. Transform DH5α E. coli competent cells with the ligation product.
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7. Inoculate the transformants (which have an ampicillin resistance gene) on the Amp+ plates to select for transformants.
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8. Colony-PCR to check which transformants contain the in-house shuttle vector + chromoprotein encoding gene.
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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.
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Part III  Transformation chromoprotein constructs and expression in Aspergillus niger
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1. Transform protoplasts from Aspergillus niger N593 with plasmids containing chromoprotein encoding genes, as well as codon-optimized chromoprotein encoding genes.
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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.
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3. Culture the A. Niger transformants and check the colour of their mycelium.
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4. Isolate fungus DNA from the mycelium and do PCR to validate insertion of the chromoprotein encoding gene.
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Part IV  Standardization of measurement of chromoprotein encoding gene induced colorization
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1. Plate DH5α containing no plasmid, containing aeBlue and eforRed in shuttle vector, eforRed and aeBlue in pSCB3 plasmid.
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2. Create ImageJ macro to standardize measurement of colorization. Useful for when the color is non-visible to the naked eye.
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Appendix
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1. information of the plasmids with chromoprotein genes
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pSB1C3-J23100-B0032-amilGFP  CDS (green) chromoprotein gene
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BBa_K592010: http://parts.igem.org/Part:BBa_K592010
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pSB1C3-J23100-B0032-eforRed  CDS (green) chromoprotein gene
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BBa_K592012: http://parts.igem.org/Part:BBa_K592012
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pSB1C3-J23100-B0032-aeBlue  CDS (green) chromoprotein gene
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BBa_K864401: http://parts.igem.org/Part:BBa_K864401
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pSB1C3-J23100-B0032-cjBlue  CDS (green) chromoprotein gene
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BBa_K592011: http://parts.igem.org/Part:BBa_K592011
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mRFP  CDS (green) chromoprotein gene
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BBa_E1010: http://parts.igem.org/wiki/index.php/Part:BBa_E1010
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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).
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Reference
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1. Alieva, N.O., et al., Diversity and evolution of coral fluorescent proteins. Plos One, 2008. 3(7): p. e2680.
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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.
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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.
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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.
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5. Shkrob, M., et al., Far-red fluorescent proteins evolved from a blue chromoprotein from Actinia equina. Biochem. J, 2005. 392: p. 649-654.
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6. Chalfie, M. and S.R. Kain, Methods of Biochemical Analysis, Green Fluorescent Protein: Properties, Applications and Protocols2005: Wiley-Liss.
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== References ==
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<span id="ref1" class="ref">1.</span>Alieva, N.O., et al., Diversity and evolution of coral fluorescent proteins. Plos One, 2008. 3(7): p. e2680.<br/>
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<span id="ref2" class="ref">2.</span>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.<br/>
 +
<span id="ref3" class="ref">3.</span>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.<br/>
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<span id="ref4" class="ref">4.</span>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.<br/>
 +
<span id="ref5" class="ref">5.</span>Shkrob, M., et al., Far-red fluorescent proteins evolved from a blue chromoprotein from Actinia equina. Biochem. J, 2005. 392: p. 649-654.<br/>
 +
<span id="ref6" class="ref">6.</span>Chalfie, M. and S.R. Kain, Methods of Biochemical Analysis, Green Fluorescent Protein: Properties, Applications and Protocols2005: Wiley-Liss.<br/>
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Latest revision as of 02:15, 5 October 2013

Chromoproteins

Simple bioreporters in synthetic biology

Outline

We aim to establish Aspergillus niger as a promising host in synthetic biology and in iGEM in particular. Therefore we introduce a range of simple bio-reporters such as pH, ATP sensors and chromoproteins, to increase the usability of the Aspergillus toolbox. Here, we focus on chromoproteins which are pigments obtained from corals. 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 fungi.

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. They 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 intrinsic colour which is visible to the naked eye. This feature, as well as their relatively small gene size, make chromoproteins simple but favorable 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 color; 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). Furthermore, far-red fluorescent proteins can 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 their ability of penetrating mammalian tissue via near-infrared light.

In addition, mutagenesis or amino acid substitutions in chromoproteins can lead to a huge increase (several hundred-fold) in their quantum yield and can influence colourization [4]. Chromophores in chromoproteins are usually characterized by a non-planar trans-conformation [5]. However, the differences in chromoprotein colour are thought to occur due to interactions between their 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

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 encoding genes aeBlue, eforRed and amilGFP respectively and DH5α E. coli strain containing mRFP were obtained from Braunschweig UR and Uppsala UR. Then the palettes of those chromoprotein transcriptional units were tested for applicability in Aspergillus niger. Besides, we synthesized and then Gibson assembled a chromoprotein encoding gene (eforRed) which was codon-optimized for Aspergillus niger. A mitochondrial retention signal was added to this codon-optimized chromoprotein encoding gene for targeted organelle localization. Afterwards protoplasts were transformed with all chromoprotein constructs, normal and codon optimized, in order to be expressed in A. niger N593.

Results

The aeBlue, amilGFP, mRFP and eforRed chromoprotein genes were succesfully inserted into an in-house vector. After their sequence was confirmed by single read sequencing, protoplasts were transformed with these chromoprotein encoding genes. Then they were inoculated on complete medium plates without uridine supplement. We thereby created selective pressure since the A. niger N593 strain owns a uridine autroxophy gene. However, unlike the E. coli transformants (Fig.2) the Aspergillus transformant colonies (Fig.3) did not show colour.

However, when performing PCR with the fungal genomic DNA as a template, the products appeared to be of the right size, giving the expected bands which were around 700bp after gel electrophoresis, see Fig 4.

The eforRed chromoprotein encoding gene was codon-optimized for A. niger and synthetic construct blocks were ordered and fused via Gibson Assembly. This synthetic construct contains a removable mitochondrial targeting sequence. The colony-PCR of E. coli transformed with this codon-optimized eforRed with mitochondrial retention sequence displayed the expected bands after gel electrophoresis, which were around 750 bp, see Fig.5. Afterwards, Aspergillus niger was transformed with the codon-optimized construct with mitochondrial retention signal. Today (05-10-2013) we noticed that the mycelium of one of the aforementioned transformed A. niger showed red colour (Fig.6)!!! The day before the wiki-freeze, we got the result we were longing for for so long!

We performed PCR on our construct in fungal DNA and on our construct in the biobrick backbone as a positive control this very evening before the wiki-freeze. It showed faint bands of the expected size (750 bp)(Fig.7)!
Lane 1 = marker, lane 2+3 = transformant fungal DNA, lane 5 = negative control, lane 7+8 = positive control (biobrick)

Fig. 7 PCR of transformant A. niger

Conclusions

The correct insertion of the non-coding optimized chromoprotein encoding genes in the fungus genomic DNA was confirmed by PCR. To further confirm whether the sequence of the chromoprotein encoding genes also remained unchanged, we sent the new constructs out for sequencing. Comparing the sequencing results to the original sequence from parts registry, for the aeBlue, mRFP and amilGFP chromoprotein gene, the identity was almost 100%. We thereby showed that the palettes of chromoprotein transcriptional units for A. niger were designed and transformed successfully. Additionally, we optimized the codon of eforRed chromoprotein encoding gene for A. niger and introduced mitochondrial retention signal for targeted localization. The transformation of A. niger with the optimized constructs was successfully validated via PCR and its mycelium showed the red colour as we expected. For the PCR however the bands for amplified transformant fungal DNA were not really intense compared to the bands for the amplified positive controls. The reason for this could be that our biobricking primers only partly annealed to our codon-optimized construct with signal sequence in the fungal DNA, while they fully anneal to our construct in biobrick vector.

To conclude, the codon optimized eforRed chromoprotein could be introduced into A. niger and applied as a simple selective marker or bioreporter. For the others whose mycelium didn’t show colour, we assume that it is either related to transcription or translation of the chromoprotein encoding gene in A. niger. Future analysis, such as RT-PCR is suggested to check if the mRNA is transcribed properly. A good alternative would be to add a His tag, purify the chromoprotein and thereby determine translation.

References

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.