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<p><h2>Abstract</h2>
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<h2>Abstract</h2>
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The UNSW iGEM team designed a new IaaH encoding part for improved functionality of the indole-3-aectamide hydrolase (IaaH) enzyme. Improving on the BBa_K1789001 part submitted by the 2015 NUDT_China team, a HisTag and GSG linker were added to the N-terminus of the enzyme, and a SpyTag and GSG linker was added to the C-terminus. Following these additions, the team was able to successfully express and purify the new part utilising the HisTag's affinity for nickel ions to purify the enzyme using IMAC purification. We also demonstrated the ability of IaaH with SpyTag to covalently bind with SpyCatcher proteins through SDS-PAGE. More details can be found on the improved part's registry page at <a target="_blank" href="http://parts.igem.org/Part:BBa_K2710005">BBa_K2710005</a>. </p>
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<p>The UNSW iGEM team designed a new IaaH encoding part for improved functionality of the indole-3-aectamide hydrolase (IaaH) enzyme. Improving on the <a target="_blank" href="http://parts.igem.org/Part:BBa_K1789001">BBa_K89001</a> part submitted by the 2015 NUDT_China team, a HisTag and GSG linker were added to the N-terminus of the enzyme, and a SpyTag and GSG linker was added to the C-terminus. Following these additions, the team was able to successfully express and purify the new part utilising the HisTag's affinity for nickel ions to purify the enzyme using IMAC purification. We also demonstrated the ability of IaaH with SpyTag to covalently bind with SpyCatcher proteins through SDS-PAGE. More details can be found on the improved part's registry page at <a target="_blank" href="http://parts.igem.org/Part:BBa_K2710005">BBa_K2710005</a>. Through these improvements, we have expanded the utility of this part twofold. First, the protein can be easily purified from cell lysates, and secondly, it now includes an irreversible attachment mechanism, robust over a range of conditions. </p>
 
</div>
 
</div>
  
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<h2>Introduction</h2>
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<h2>The Enzyme</h2>
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<p>The components of the prefoldin scaffold and the associated attached enzymes were to be expressed for enzymatic and self-assembly experiments. To produce the proteins required for these analytical experiments, our gene of interests were to be cloned into an appropriate plasmid vector for expression and purification in <i>E. coli</i> cells. This page describes the methods undertaken to produce recombinant plasmids containing our DNA constructs for protein expression. Eight codon optimised DNA constructs were designed and synthesised in the form of g-Blocks from Integrated DNA Technologies (IDT).</p>
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<p> <b>Indole-3-acetamide hydrolase (IaaH)</b> is an enzyme involved in the biosynthesis of indole-3-aectic acid. IaaH originating from <i>Alcaligenes sp.</i> strain HPC127114 was fused with a SpyTag and a hexahistidine tag (HisTag)<sup>1</sup>. </p>
  
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<p>The hetero-hexameric structure of our scaffold is composed of two alpha prefoldin (aPFD) subunits and four beta prefoldin (bPFD) subunits covalently attached to enzymes<sup><a href=#references>2</a></sup>. To produce a functional and complete scaffold, genes encoding the proteins which form the scaffold must first be cloned into an appropriate vector. Plasmids containing aPFD and bPFD were constructed. In addition, aPFD-SpyCatcher and bPFD-SnoopCatcher fusion constructs were created for comparative enzyme activity experiments.</p>
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<p>The auxin indole-3-acetic acid, is a plant hormone involved in the regulation of plant growth and development<sup>2</sup>. Indole-3-aecetic acid can be synthesised via the indole-3-acetamide pathway, which converts tryptophan to indole-3-aectic acid in a two-step enzymatic pathway<sup>3</sup> <b>(Figure 1)</b>. The flavoprotein tryptophan 2-monooxygenase (IaaM) catalyses the oxidative decarboxylation of tryptophan to indole-3-acetamide in the first, rate limiting step of the pathway<sup>4</sup>. Subsequently, the enzyme indole-3-acetamide hydrolase (IaaH) converts indole-3-acetamide to indole-3-aectic acid<sup>1</sup>.</p>
  
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<p>SpyTags<sup><a href=#references>3</a></sup>and SnoopTags<sup><a href=#references>3</a></sup> were fused to indole-3-acetamide hydrolase (IaaH-SpyTag) and tryptophan 2-monoxygenase (IaaM-SnoopTag) and cloned into appropriate plasmid vectors. Expression of these plasmids would enable protein conjugation experiments with the prefoldin-catcher protein constructs. Fluorescent mCerulean3-SnoopTag and mVenus-SpyTag gBlocks were also cloned into plasmids for Försters Resonance Energy Transfer (FRET) experiments. 6x His-Tags were also attached to the start of each DNA construct via a GSG linker, which would allow the recovery of purified protein.</p>
 
  
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<p>All original gBlock designs contain the BioBrick prefix and suffix sequences which were flanked by 20-25 bp long 5’ and 3’ Gibson overhangs. Cloning the BioBrick restriction sites into the DNA construct allowed excision of the insert out from the plasmid vector for diagnostic purposes and for transfer of the inserts into the pSB1C3 BioBrick backbone. Gibson Assembly cloning techniques were used to clone the DNA constructs into the plasmids. 5’ exonuclease activity generates complementary overhang sequences on the insert and vector, and polymerase fills in the gaps of the single strand regions. DNA ligase seals the nicks of the gaps, allowing the two fragments to covalently link together (Figure 1)<sup><a href=#references>1</a></sup>.<p>
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<div class=image-box>
 
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<img src=https://static.igem.org/mediawiki/2018/5/52/T--UNSW_Australia--cloning-img10-gibson.png>
 
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</div>
 
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<p class=figure-legend><b>Figure 1:</b> Diagram illustrating the process of Gibson assembly sequence insertion into the plasmid vector<sup><a href=#references>1</a></sup>.</p>
 
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<p>Our DNA constructs were cloned into pETDuet-1 and pRSFDuet-1 plasmid vectors, as well as pET-19b in our later experiments. The Duet vectors carry two expression units that are controlled by a T7-lac promoter and terminator for protein expression. The Duet plasmids, pETDuet-1 and pPRSFDuet-1, both possess an ampicillin and kanamycin resistance gene, respectively. Meanwhile pET-19 confers ampicillin resistance (Figure 2). These specific vectors were chosen so that the prefoldin-catcher and enzyme-tag DNA constructs could be cloned into the same cell, allowing the entire scaffold to be expressed simultaneously. Furthermore, pETDuet-1 and pRSFDuet-1 plasmids possess different origins of replication,  which enables in vivo production of the scaffold-enzyme complex.</p>
 
  
 
<div class=image-box>
 
<div class=image-box>
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<img src=https://static.igem.org/mediawiki/2018/5/59/T--UNSW_Australia--cloning-img2-plasmids.png>
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<img src=https://static.igem.org/mediawiki/2018/7/78/T--UNSW_Australia--bec-design-iaa-pathway.png>
 
</div>
 
</div>
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<p class=figure-legend><b>Figure 2:</b> Plasmid maps depicting pETDuet-1, pRSFDuet-1 and pET19-b. Resistance genes are shown in red. Images were generated by Benchling.</p>
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<p class=figure-legend><b>Figure 1:</b> The indole-3-acetamide pathway for indole-3-aecetic acid biosynthesis.</p>
  
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<p>We successfully cloned 8 DNA constructs were successfully cloned into pET-Duet1 and pRSF-Duet1 plasmids. However, we experienced difficulties expressing protein from these plasmids. The constructs were then redesigned to omit the iGEM prefix and suffix sequences and the Gibson overhangs were modified. This enabled the cloning of 6 modified constructs into the pET-19b vector for protein expression and purification. We opted to switch to this vector because it was currently being used successfully by a collaborator for protein expression.</p>
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<br />
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<h2>Improvements</h2>
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<p> The original part was improved by two key additions; a <b>SpyTag</b> and a <b>HisTag</b>.
  
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<p>Circular pETDuet-1 and pRSFDuet-1 and linearised pET-19b were kindly supplied by Dr Dominic Glover.</p>
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<p>The <b>SpyTag</b> forms one component of the SpyTag/SpyCatcher system, which enables covalent attachment of two proteins<sup>6</sup>. The SpyTag and SpyCatcher system was created by cleaving the CnaB2 domain of the fibronectin-binding protein FbaB derived from <i>Streptococcus pyogenes</i> to form a thirteen residue SpyTag peptide and a 116-residue SpyCatcher peptide<sup>6</sup>. The SpyTag (1.1 kDa) and SpyCatcher (12 kDa) form an irreversible intramolecular isopeptide bond between Asp<sup>117</sup> on SpyTag and Lys<sup>31</sup> on SpyCatcher<sup>6</sup>, spontaneously and specifically binding to each other so that they can be used as attachment mechanisms to create new, self-assembling protein arrangements<sup>6</sup> <b>(Figure 2)</b>.</p>
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<p>It is particularly useful because neither component needs to be at the C or N terminus<sup>7</sup>, and the effect on the attached protein’s activity appears to be negligible<sup>8</sup>. It also reported as useful in a variety of reaction conditions, with Howarth showing that the SpyTag/SpyCatcher “had a high yield...required only simple mixing (and) tolerated diverse conditions (pH, buffer components and temperature)”<sup>9</sup>. </p>
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<p class=table-legend><b>Table 1:</b> The constructs to be cloned into each vector.</p>
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<div class="flex-center">
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<table class="lab-table">
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<tr>
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<th>pETDuet-1</th>
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<th>pRSFDuet-1</th>
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<th>pET-19b</th>
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</tr>
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<tr>
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<td>aPFD</td>
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<td>IaaH-SpyTag</td>
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<td>aPFD</td>
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</tr>
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<tr>
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<td>bPFD</td>
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<td>IaaM-SnoopTag</td>
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<td>bPFD</td>
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</tr>
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<tr>
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<td>aPFD-SpyCatcher</td>
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<td>mCerulean3-SnoopTag</td>
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<td>IaaH-SpyTag</td>
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</tr>
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<tr>
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<td>bPFD-SnoopCatcher</td>
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<td>mVenus-SpyTag</td>
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<td>IaaM-SnoopTag</td>
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</tr>
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<tr>
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<td></td>
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<td></td>
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<td>IaaH</td>
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</tr>
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<tr>
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<td></td>
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<td></td>
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<td>IaaM</td>
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</tr>
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</table>
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</div>
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<h2>Aim</h2>
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<p>To clone genes encoding the parts required to form our scaffold into appropriate plasmid vectors by Gibson assembly. In particular:</p>
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<ol>
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<li>Cloning original DNA constructs into pETDuet-1 and pRSFDuet-1</li>
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<li>Cloning modified DNA constructs into pET-19b</li>
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</ol>
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<h2>DNA design</h2>
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<p>We designed 8 gBlocks for cloning by Gibson assembly into the first multiple cloning sites of pETDuet-1 and pRSFDuet-1 (Figure 3). All sequences included an N-terminal 6xHis-Tag immediately after the start codon to enable purification using Nickel affinity, followed by a Glycine-Serine-Glycine (GSG) linker. The GSG linker provides flexibility as the side chains of glycine and serine are small, and can allow the 6xHis-Tag to move freely in solution. The amino acid sequences for each protein were obtained. For prefoldin-catcher fusion proteins, a GSGSGSGSG linker and SpyCatcher or SnoopCatcher followed the alpha or beta prefoldin sequence, yielding aPFD-SpyCatcher and bPFD-SnoopCatcher. For enzyme-tag fusion proteins, a GSG linker and SpyTag or SnoopTag followed the enzyme sequence, yielding IaaH-SpyTag and IaaM-SnoopTag. The longer 9 amino acid linker was used for SpyCatcher and SnoopCatcher fusion proteins as the catcher domains are large and may sterically interfere with protein folding if the C-terminus of the original protein is not solvent accessible. An increased linker length on the aPFD-SpyCatcher and bPFD-SnoopCatcher fusions may also enable the scaffold to accommodate the attachment of large enzymes. The DNA sequences were codon optimised for <i>E. coli</i> with manual removal of EcoRI, XbaI, SpeI and PstI restriction sites for RFC10 compatibility. The BioBrick prefix and suffix were then placed on the 5’ and 3’ ends of each sequence. Finally, the DNA sequences were flanked with 25 bp Gibson overhangs identical to the 25 bp immediately upstream and downstream of the insertion site into pETDuet-1 and pRSFDuet-1.</p>
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<div class=image-box>
 
<div class=image-box>
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<img src=https://static.igem.org/mediawiki/2018/8/83/T--UNSW_Australia--cloning-img3-gblock.png>
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<img src=https://static.igem.org/mediawiki/2018/8/82/T--UNSW_Australia--bec-design-spytc.png>
 
</div>
 
</div>
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<p class=figure-legend><b>Figure 2:</b> A spontaneous isopeptide bond forms between SpyTag and SpyCatcher. Image created using PDB ID: 4MLS<sup>10</sup> </p>
  
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<p class=figure-legend><b>Figure 3:</b> DNA constructs designed by Brian Ee. Images were generated by Benchling.</p>
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<br />
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<p>A <b>HisTag</b> (six consecutive histidine residues, also known as a hexahistidine tag) was added to IaaH to enable purification, utilising the affinity of the HisTag for nickel ions for Immobilised Metal Affinity Chromatography purification<sup>5</sup>.</p>
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  <br />
  
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<h2>Method</h2>
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<h2>Experimental Characterisation</h2>
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<p>pETDuet-1 and pRSFDuet-1 plasmids were linearised with PCR (NEB), removing the first multiple cloning site. Enzymes were removed by PCR clean up (Sigma Aldrich). A DpnI digest was performed to remove template circular plasmid and the product was then cleaned up again. Linearity was confirmed by agarose gel electrophoresis. Gibson assembly was used to construct plasmids by combining linearised plasmids with gBlocks ordered from IDT. The Gibson assembly product was transformed via heat shock into <i>E. coli</I> DH5-alpha (NEB) cells before plating onto antibiotic-selective Luria broth agar plates. A colony PCR was performed and analysed by gel electrophoresis in order to identify single colonies that had been successfully transformed with Gibson assembly products. Successful transformants were grown in 10 mL of LB with appropriate antibiotic and plasmids were prepared using QIAprep Spin Miniprep Kit (Qiagen). To confirm the insertion of our DNA construct in the miniprepped plasmids, we performed a diagnostic digest. Plasmids were digested with restriction enzymes that excised the insert and analysed with agarose gel electrophoresis. Sanger sequencing was also performed (Ramaciotti Centre for Genomics) to verify gene sequences.</p>
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<p> The improved part was validated by sequence verification and a diagnostic gel.  Following this, the original and improved part were cloned into the expression vector pET19B and successfully expressed in <i>E. coli</i> T7 Express cells (NEB). The HisTag and SpyTag improvements were experimentally characterised through SDS-PAGE analysis of IMAC purifications and attachment to SpyCatcher fusion proteins. Refer to the <a target="_blank" href="http://parts.igem.org/Part:BBa_K2710005">BBa_K2710005</a> registry page for details of the experimental characterisation.</p>
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<p>For the transfer of inserts into pET-19b, primers were designed to PCR amplify the inserts whilst also removing the BioBrick prefix and suffix, and adding Gibson overhangs appropriate for insertion into the multiple cloning site of pET-19b. Gibson assembly was then performed as previously described with PCR linearised pET-19b.</p>
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<p>For more detail on our cloning protocols, visit our <a href=”https://2018.igem.org/Team:UNSW_Australia/Experiments”>experiment page</a></p>
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<p>Through these improvements, we have expanded the utility of this part twofold. First, the protein can be easily purified from cell lysates, and secondly, it now includes an irreversible attachment mechanism, robust over a range of conditions. </p>
  
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<div class=image-box>
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<p>Furthermore, as wild-type gPFD polymerises to form filamentous structures, Transmission Electron Microscopy (TEM) was performed on gPFD-SpyC attached to IaaH-SpyT to investigate the effect of the attachment of the enzyme on filament formation. Attachment via the SpyTag/SpyCatcher mechanism appears to distort the filaments <b>(Figure 3)</b>.</p>
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<img src=https://static.igem.org/mediawiki/2018/b/b0/T--UNSW_Australia--cloning-img4-cloningflowchart.png>
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</div>
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<p class=figure-legend><b>Figure 4:</b> Flowchart depicting the overall cloning process undertaken.</p>
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<div id=results>
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<h2>Results</h2>
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<h3>Cloning original DNA constructs into pETDuet-1 and pRSFDuet-1</h3>
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<p>All 8 original DNA constructs (Figure 3) were successfully cloned into pET-Duet1 and pRSF-Duet1 plasmid vectors. The presence of each inserted gene within its plasmid was confirmed by performing a diagnostic digest (Figure 5), and the recombinant plasmids were also each sequence verified. These plasmids were used for our initial attempts at protein expression and purification.</p>
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<div class=image-box>
 
<div class=image-box>
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<img src=https://static.igem.org/mediawiki/2018/c/c2/T--UNSW_Australia--cloning-img5-gel1.png>
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<img src=https://static.igem.org/mediawiki/2018/thumb/f/f5/T--UNSW_Australia--TEM.jpeg/800px-T--UNSW_Australia--TEM.jpeg>
 
</div>
 
</div>
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<p class=figure-legend><b>Figure 5:</b> Diagnostic digest of recombinant pETDuet-1 and pRSFDuet-1 plasmids. Gel demonstrates the construction of plasmids containing the desired DNA inserts. Plasmids were restriction enzyme digested with EcoRI and PstI and analysed by agarose gel electrophoresis. The red boxes indicate the presence of the insert at the expected size in comparison to the 2-Log DNA marker.</p>
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<p class="figure-legend fig5-leg"><b>Figure 3: </b>TEM demonstrates that gPFD and gPFD mutants are able to assemble and form filaments. A: Wild-type gPFD filaments. B: gPFD-SpyC filaments, indicating that gPFD is able to form filaments when fused to a SpyCatcher. C: gPFD-SpyC reacted with IaaH-SpyT. Clumps of filaments, or curled filaments were observed.</p>
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<h3>Cloning modified DNA constructs into pET-19b</h3>
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<p>The eight recombinant pETDuet-1 and pRSFDuet-1 recombinant plasmids were unable to be expressed, so six DNA constructs were modified and cloned into the pET-19b vector instead. The presence of aPFD, bPFD and IaaH-SpyTag in pET-19b was confirmed by a diagnostic digest (Figure 6). Moreover, Sanger sequencing verified successful insertion of these genes as well as aPFD-SpyCatcher, bPFD-SnoopCatcher and IaaM-SnoopTag. Therefore, the following modified constructs were successfully cloned into pET-19b:</p>
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<ul>
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<li>aPFD</li>
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<li>bPFD</li>
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<li>aPFD-SpyCatcher</li>
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<li>bPFD-SnoopCatcher</li>
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<li>IaaH-SpyTag</li>
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<li>IaaM-SnoopTag</li>
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</ul>
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<p>In addition, the following BioBricks were obtained from the iGEM distribution plates and cloned into pET-19b for creation of our <a href=https://2018.igem.org/Team:UNSW_Australia/Improve target=_blank>Improved Part</a>.<p>
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<ul>
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<li>IaaM <a target=_blank href=http://parts.igem.org/Part:BBa_K1789000>(BBa_K1789000)</a></li>
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<li>IaaH <a target=_blank href=http://parts.igem.org/Part:BBa_K1789001>(BBa_K1789001)</a></li>
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</ul>
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</div>
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<div class=image-box>
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<img src=https://static.igem.org/mediawiki/2018/c/cc/T--UNSW_Australia--cloning-img6-gel2.png>
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</div>
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<p class=figure-legend><b>Figure 6:</b> Diagnostic digest of recombinant pET-19b plasmids. Gel demonstrates the construction of recombinant pET-19b plasmids containing aPFD, bPFD and IaaH-SpyT. Plasmids were restriction enzyme digested with EcoRI and XbaI and analysed by agarose gel electrophoresis. The red boxes indicate the presence of the insert at the expected size in comparison to the 2-Log DNA marker. </p>
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<h2>Discussion</h2>
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<p>The initial aim of cloning all 8 DNA constructs into pETDuet-1 and pRSFDuet-1 plasmid vectors was successful. We originally opted for these two Duet vectors as it would enable us to co-transform and express two target genes into MCS1 and MCS2 if time permitted. This could have streamlined the process for hetero-hexameric complex assembly, increasing the efficiency of future cloning experiments.
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However, we were unable to express our target proteins when the recombinant plasmids were transformed into expression strains for protein purification experiments. We hypothesised that this was due to the 20 bp long BioBrick prefix situated between the ribosomal binding site (RBS) and the start codon of our construct. This displaces the RBS away from the start of transcription, which is the likely cause for the difficulties experienced with protein expression. Translation studies in E. coli have demonstrated that the optimal spacing between the RBS and the start codon in E. coli ranges from 7-9 nucleotides<sup><a href=#references>4</a></sup>.</p>
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<p>We decided to clone the DNA constructs into the pET-19b vector as it was used successfully in previous cloning experiments for our collaborators. Dr Dominic Glover kindly supplied us with linearised pET-19b plasmids for our experiments, and we were successful in cloning 6 of our 8 DNA constructs into these vectors. Unfortunately due to time restrictions, we were unable to clone all 8 DNA constructs in pET-19b. In the future, we hope to clone more parts into pET-19b which would enable us to perform more assembly tests and ultimately piece together and characterise a complete and functional scaffold.</p>
 
  
 
<div id=references>
 
<div id=references>
 
<h2>References</h2>
 
<h2>References</h2>
 
<ol>
 
<ol>
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<li>Gibson, D. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. <i>Nature Methods</i> 6, 343-345 (2009).</li>
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<li>Mishra, P., Kaur, S., Sharma, A. N. and Jolly, R. S. Characterization of an Indole-3-Acetamide Hydrolase from Alcaligenes faecalis subsp. parafaecalis and Its Application in Efficient Preparation of Both Enantiomers of Chiral Building Block 2,3-Dihydro-1,4-Benzodioxin-2-Carboxylic Acid. <i>PLoS One 11</i>, e0159009 (2016).</li>
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<li>Siegert, R., Leroux, M., Scheufler, C., Hartl, F. & Moarefi, I. Structure of the Molecular Chaperone Prefoldin. <i>Cell</i> 103, 621-632 (2000).</li>
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<li>Davies, P. J. Plant Hormones: Biosynthesis, Signal Transduction, Action! ,  (Springer Netherlands, 2007).</li>
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<li>Zakeri, B. et al. Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. <i>Proceedings of the National Academy of Sciences</i> 109, E690-E697 (2012).</li>
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<li>Spaepen, S., Vanderleyden, J. and Remans, R. Indole-3-acetic acid in microbial and microorganism-plant signaling. <i>FEMS Microbiol Rev</i>, <b>31</b> 425-448, doi:10.1111/j.1574-6976.2007.00072.x (2007).</li>
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<li>Vellanoweth, R. & Rabinowitz, J. The influence of ribosome-binding-site elements on translational efficiency in Bacillus subtilis and <i>Escherichia coli</i> in vivo. <i>Molecular Microbiology</i> 6, 1105-1114 (1992).</li>
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<li>Gaweska, H. M., Taylor, A. B., Hart, P. J. and Fitzpatrick, P. F. Structure of the flavoprotein tryptophan 2-monooxygenase, a key enzyme in the formation of galls in plants. <em>Biochemistry</em>. <b>52</b> 2620–6 (2013).</li>
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<li>Hochuli, E., Dobeli, H. and Schacher, A. New metal chelate adsorbent selective for proteins and peptides containing neighbouring histidine residues. <i>J Chromatogr.</i> <b>411</b> 177-184 (1987).</li>
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<li>Zakeri, B. et al. Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. <i>Proc Natl Acad Sci U S A.</i> <b>109</b> E690-697, doi:10.1073/pnas.1115485109 (2012).</li>
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<li>Domeradzka, N. E., Werten, M. W., Wolf, F. A. and de Vries, R. Protein cross-linking tools for the construction of nanomaterials. <i>Curr Opin Biotechnol.</i> <b>39</b> 61-67, doi:10.1016/j.copbio.2016.01.003 (2016).</li>
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<li>Walper, S. A., Turner, K. B. and Medintz, I. L. Enzymatic bioconjugation of nanoparticles: developing specificity and control. <i>Curr Opin Biotechnol.</i> <b>34</b> 232-241, doi:10.1016/j.copbio.2015.04.003 (2015).</li>
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<li>Reddington, S. C. and Howarth, M. Secrets of a covalent interaction for biomaterials and biotechnology: SpyTag and SpyCatcher. <i>Curr Opin Chem Biol.</i> <b>29</b> 94-99, doi:10.1016/j.cbpa.2015.10.002 (2015).</li>
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<li>Li, L., Fierer, J. O., Rapoport, T. A. and Howarth, M. Structural Analysis and Optimization of the Covalent Association between SpyCatcher and a Peptide Tag. <i>J. Mol. Biol.</i> <b>426</b> 309–317 (2014).</li>
 
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Latest revision as of 03:30, 18 October 2018

Improved Part

Abstract

The UNSW iGEM team designed a new IaaH encoding part for improved functionality of the indole-3-aectamide hydrolase (IaaH) enzyme. Improving on the BBa_K89001 part submitted by the 2015 NUDT_China team, a HisTag and GSG linker were added to the N-terminus of the enzyme, and a SpyTag and GSG linker was added to the C-terminus. Following these additions, the team was able to successfully express and purify the new part utilising the HisTag's affinity for nickel ions to purify the enzyme using IMAC purification. We also demonstrated the ability of IaaH with SpyTag to covalently bind with SpyCatcher proteins through SDS-PAGE. More details can be found on the improved part's registry page at BBa_K2710005. Through these improvements, we have expanded the utility of this part twofold. First, the protein can be easily purified from cell lysates, and secondly, it now includes an irreversible attachment mechanism, robust over a range of conditions.

The Enzyme

Indole-3-acetamide hydrolase (IaaH) is an enzyme involved in the biosynthesis of indole-3-aectic acid. IaaH originating from Alcaligenes sp. strain HPC127114 was fused with a SpyTag and a hexahistidine tag (HisTag)1.

The auxin indole-3-acetic acid, is a plant hormone involved in the regulation of plant growth and development2. Indole-3-aecetic acid can be synthesised via the indole-3-acetamide pathway, which converts tryptophan to indole-3-aectic acid in a two-step enzymatic pathway3 (Figure 1). The flavoprotein tryptophan 2-monooxygenase (IaaM) catalyses the oxidative decarboxylation of tryptophan to indole-3-acetamide in the first, rate limiting step of the pathway4. Subsequently, the enzyme indole-3-acetamide hydrolase (IaaH) converts indole-3-acetamide to indole-3-aectic acid1.


Figure 1: The indole-3-acetamide pathway for indole-3-aecetic acid biosynthesis.


Improvements

The original part was improved by two key additions; a SpyTag and a HisTag.

The SpyTag forms one component of the SpyTag/SpyCatcher system, which enables covalent attachment of two proteins6. The SpyTag and SpyCatcher system was created by cleaving the CnaB2 domain of the fibronectin-binding protein FbaB derived from Streptococcus pyogenes to form a thirteen residue SpyTag peptide and a 116-residue SpyCatcher peptide6. The SpyTag (1.1 kDa) and SpyCatcher (12 kDa) form an irreversible intramolecular isopeptide bond between Asp117 on SpyTag and Lys31 on SpyCatcher6, spontaneously and specifically binding to each other so that they can be used as attachment mechanisms to create new, self-assembling protein arrangements6 (Figure 2).

It is particularly useful because neither component needs to be at the C or N terminus7, and the effect on the attached protein’s activity appears to be negligible8. It also reported as useful in a variety of reaction conditions, with Howarth showing that the SpyTag/SpyCatcher “had a high yield...required only simple mixing (and) tolerated diverse conditions (pH, buffer components and temperature)”9.

Figure 2: A spontaneous isopeptide bond forms between SpyTag and SpyCatcher. Image created using PDB ID: 4MLS10


A HisTag (six consecutive histidine residues, also known as a hexahistidine tag) was added to IaaH to enable purification, utilising the affinity of the HisTag for nickel ions for Immobilised Metal Affinity Chromatography purification5.


Experimental Characterisation

The improved part was validated by sequence verification and a diagnostic gel. Following this, the original and improved part were cloned into the expression vector pET19B and successfully expressed in E. coli T7 Express cells (NEB). The HisTag and SpyTag improvements were experimentally characterised through SDS-PAGE analysis of IMAC purifications and attachment to SpyCatcher fusion proteins. Refer to the BBa_K2710005 registry page for details of the experimental characterisation.

Through these improvements, we have expanded the utility of this part twofold. First, the protein can be easily purified from cell lysates, and secondly, it now includes an irreversible attachment mechanism, robust over a range of conditions.

Furthermore, as wild-type gPFD polymerises to form filamentous structures, Transmission Electron Microscopy (TEM) was performed on gPFD-SpyC attached to IaaH-SpyT to investigate the effect of the attachment of the enzyme on filament formation. Attachment via the SpyTag/SpyCatcher mechanism appears to distort the filaments (Figure 3).


Figure 3: TEM demonstrates that gPFD and gPFD mutants are able to assemble and form filaments. A: Wild-type gPFD filaments. B: gPFD-SpyC filaments, indicating that gPFD is able to form filaments when fused to a SpyCatcher. C: gPFD-SpyC reacted with IaaH-SpyT. Clumps of filaments, or curled filaments were observed.

References

  1. Mishra, P., Kaur, S., Sharma, A. N. and Jolly, R. S. Characterization of an Indole-3-Acetamide Hydrolase from Alcaligenes faecalis subsp. parafaecalis and Its Application in Efficient Preparation of Both Enantiomers of Chiral Building Block 2,3-Dihydro-1,4-Benzodioxin-2-Carboxylic Acid. PLoS One 11, e0159009 (2016).
  2. Davies, P. J. Plant Hormones: Biosynthesis, Signal Transduction, Action! , (Springer Netherlands, 2007).
  3. Spaepen, S., Vanderleyden, J. and Remans, R. Indole-3-acetic acid in microbial and microorganism-plant signaling. FEMS Microbiol Rev, 31 425-448, doi:10.1111/j.1574-6976.2007.00072.x (2007).
  4. Gaweska, H. M., Taylor, A. B., Hart, P. J. and Fitzpatrick, P. F. Structure of the flavoprotein tryptophan 2-monooxygenase, a key enzyme in the formation of galls in plants. Biochemistry. 52 2620–6 (2013).
  5. Hochuli, E., Dobeli, H. and Schacher, A. New metal chelate adsorbent selective for proteins and peptides containing neighbouring histidine residues. J Chromatogr. 411 177-184 (1987).
  6. Zakeri, B. et al. Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proc Natl Acad Sci U S A. 109 E690-697, doi:10.1073/pnas.1115485109 (2012).
  7. Domeradzka, N. E., Werten, M. W., Wolf, F. A. and de Vries, R. Protein cross-linking tools for the construction of nanomaterials. Curr Opin Biotechnol. 39 61-67, doi:10.1016/j.copbio.2016.01.003 (2016).
  8. Walper, S. A., Turner, K. B. and Medintz, I. L. Enzymatic bioconjugation of nanoparticles: developing specificity and control. Curr Opin Biotechnol. 34 232-241, doi:10.1016/j.copbio.2015.04.003 (2015).
  9. Reddington, S. C. and Howarth, M. Secrets of a covalent interaction for biomaterials and biotechnology: SpyTag and SpyCatcher. Curr Opin Chem Biol. 29 94-99, doi:10.1016/j.cbpa.2015.10.002 (2015).
  10. Li, L., Fierer, J. O., Rapoport, T. A. and Howarth, M. Structural Analysis and Optimization of the Covalent Association between SpyCatcher and a Peptide Tag. J. Mol. Biol. 426 309–317 (2014).