Difference between revisions of "Team:Paris Bettencourt/Project/Enzyme"

 
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<body>
 
<body>
 
<div id=topheader> </div>
 
<div id=topheader> </div>
 
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<br>
<h1 class="red">Enzyme group: Repurposing and Enhancing Enzymes for Anthocyanin degradation  </h1>
+
  
  
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     <ul>
 
     <ul>
 
         <li>Identification of potential Anthocyanin degrading enzymes.</li>
 
         <li>Identification of potential Anthocyanin degrading enzymes.</li>
         <li>Heterologous expression of the selected Anthocyanin degrading enzymes in <i>E. coli</i>.</li>
+
         <li>Heterologous expression of the potential Anthocyanin degrading enzymes in <i>E. coli</i>.</li>
         <li>Enhancement of the enzymatic wine stain degradation through the construction of Anthocyanin degrading enzymes fused to Fabric Binding Domains <ahref="https://2016.igem.org/Team:Paris_Bettencourt/Project/Binding">More information on the FBD</a>.</li> 
+
         <li>Enhancement of the enzymatic wine stain degradation through the construction of potential Anthocyanin degrading enzymes fused to Fabric Binding Domains. More information on FBD on Binding.
 
     </ul>
 
     </ul>
 
</div>
 
</div>
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<div style="clear: both;"></div>
 
<div style="clear: both;"></div>
  
<div class="projtile2">
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<div class="projtile3">
 
     <h2 class="red" style="text-align:center;">BioBricks</h2>
 
     <h2 class="red" style="text-align:center;">BioBricks</h2>
            <ul>
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 +
        <ul>
 
<li>BBa_K2043001
 
<li>BBa_K2043001
 
<li>BBa_K2043002
 
<li>BBa_K2043002
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<li>BBa_K2043006
 
<li>BBa_K2043006
 
<li>BBa_K2043007
 
<li>BBa_K2043007
<li>BBa_K2043008
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                                <li>BBa_K2043008
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                                </li>
 +
                      </ul>
 +
</div>
 +
 
 +
<div style="clear: both;"></div>
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 +
<div class="projtile5">
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        <ul>
 
<li>BBa_K2043009
 
<li>BBa_K2043009
 
<li>BBa_K2043010
 
<li>BBa_K2043010
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<li>BBa_K2043015
 
<li>BBa_K2043015
 
<li>BBa_K2043016
 
<li>BBa_K2043016
<li>BBa_K2043017</li>
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<li>BBa_K2043017
            </ul>
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                                </li>
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                      </ul>
 
</div>
 
</div>
  
 
<div style="clear: both;"></div>
 
<div style="clear: both;"></div>
  
<div class="projtile3">
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<div class="projtile2">
 
       <h2 class="red" style="text-align:center;">Results</h2>
 
       <h2 class="red" style="text-align:center;">Results</h2>
 
       <ul>
 
       <ul>
 
             <li>Six candidate plant and bacterial enzymes were tested for expression in <i>E. coli</i>
 
             <li>Six candidate plant and bacterial enzymes were tested for expression in <i>E. coli</i>
             <li>Three out of six enzymes were functionally expressed in <i>E. coli</i> and tested for their ability to degrade Anthocyanins
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             <li>Three out of six enzymes were functionally expressed in <i>E. coli</i> and tested for their ability to degrade their natural substrate
             <li>Finally, the selected enzymes were fused with Fabric Binding Domains and tested for enhanced stain removal activity </li>
+
             <li>Finally, the several enzymes were fused with Fabric Binding Domains and their activity was still observed. FBD-GFP fusion proteins revealed affinity and specificity in our FBDs </li>
 
       </ul>
 
       </ul>
 
</div>
 
</div>
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<li>Induced protein expression by the use of expression systems
 
<li>Induced protein expression by the use of expression systems
 
<li>Preparation of cell extract for protein activity testing
 
<li>Preparation of cell extract for protein activity testing
<li>Assaying the binding strength of the Fabric Binding Domains <ahref="https://2016.igem.org/Team:Paris_Bettencourt/Project/Binding">More information on the FBD</a>
+
<li>Assaying the binding strength of the Fabric Binding Domains. More information on the FBD on Binding
 
<li>Assaying the enhanced stain removal activity of fusion proteins</li>
 
<li>Assaying the enhanced stain removal activity of fusion proteins</li>
 
     </ul>
 
     </ul>
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<div id=subheader>
 
<div id=subheader>
 
<div id="input">
 
<div id="input">
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<div style="margin-top:0px; margin-bottom:20px; margin-left:70px;">
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<div class="panel" >
 +
 +
    <a href="https://2016.igem.org/Team:Paris_Bettencourt/Project/Enzyme#ancre" title="Results">
 +
<div id="thpanel" class="subpanel3">     
 +
  <img class="narrowimg" src="https://static.igem.org/mediawiki/2016/a/a7/Paris_Bettencourt-Result_Button.jpeg" width="400px" height="80px"/>
 +
  <div class="titlebox2">
 +
  Skip to results
 +
  </div>
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</div>
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      </a>
 +
 +
      <a href="https://2016.igem.org/Team:Paris_Bettencourt/Description" title="Project description">
 +
<div id="thpanel" class="subpanel3">
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  <img class="narrowimg" src="https://static.igem.org/mediawiki/2016/2/28/Paris_Bettencourt-Project_Button.jpeg" width="400px" height="80px"/>
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  <div class="titlebox2">
 +
    Overview of the project
 +
  </div>
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</div>
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      </a>
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</div>
 +
</div>
 +
 +
 
<h2 class="red">Abstract</h2>
 
<h2 class="red">Abstract</h2>
<p>
+
<p><b>
<b>Anthocyanins, they key pigments present in wine, are polyphenol molecules naturally found in many plants. In this part of the project we turn to nature to search for enzymes able to degrade these pigments, and we take it a step further by enhancing their stain removal activity. We aim to harness the power of nature and make the changes that would direct it to our goal. First, we searched the literature for enzymes that had potential to degrade Anthocyanins. Then, making use of the Fabric Binding Domains found in this project, we directed their activity to our stains.<br> Here we show that our enzymes are correctly expressed and maintain their activity even when fused with the Fabric Binding Domains. We also show that our Fabric Binding Domains have specificity to different fabrics, opening a new door for enzymatic detergents for stain removal. SPEAK ABOUT HOW WE DEGRADE ANTHOCYANINS!!!</b>
+
Anthocyanins, the key pigments present in wine, are polyphenolic molecules that are naturally found in many plants. In this part of our iGEM project we turn to nature in the search for enzymes with the potential ability to degrade these pigments for expression in <i>E. coli</i>. Three potential wine stain removers, BpuI, CatA and XylE, were successfully expressed in <i>E. coli</i> and are ready to be tested for their ability to degrade Anthocyanins. To take their stain removal capacity to the next level, we discovered peptide binding domains that supported the localization of the enzymes to cotton, wool, linen and silk. Here we also showed that enzymes XylE and CatA maintained their activity when fused to our Fabric Binding Domains. We believe that our fabric-specific enzyme detergents have opened a new door for the development of innovative laundry products.
</p>
+
</b></p>
  
  
 
<h2 class="red"> Motivation and Background</h2>
 
<h2 class="red"> Motivation and Background</h2>
  
<h3>Nature, from inspiration to a canvas</h3>
+
<h3<Motivation</h3>
 
<p>
 
<p>
Throughout human history, people have always taken inspiration on nature for their daily problems. We find numerous examples of turning to nature in many fields. The most famous and successful example of this is probably velcro, which was inspired in the way the burrs’ structure allows them to attach to furry bodies, such as dogs. <br>Over the years, however, with the increasing control that humans have over nature, we have been allowed to go one step further. We no longer only take inspiration on nature, but also control it and change it, “enhancing” it for our specific purposes. <br>It is with this mindset that we approached this part of our project. It is clear that in the past we have turned to nature to find enzymes that can act as specific detergents in our laundry. We wanted to do the same, to find specific enzymes for getting rid of wine stains, but we wanted to go a step further. We wanted to enhance the stain removal capacity of these enzymes by focusing their activity on our stains.
+
Throughout human history, people have taken inspiration from nature to find solutions for everyday problems. A famous example is velcro. Velcro was inspired by the way the structure of plant burrs allowed them to attach to the furry bodies of animals. <br>
 +
As technology has progressed, we not only take inspiration from nature, but also control and enhance biology for our specific purposes. <br>
 +
We adopted this mindset when we approached this part of our project: the development of anthocyanin degrading enzymes that can be attached to a specific type of fabric.<br>
 +
First, we searched for enzyme candidates that could be suitable within this scope. We selected enzymes from literature based on the following three criteria:<br>
 +
1- the enzyme has the known ability to specifically degrade Anthocyanins, or, more broadly, polyphenols, which are the class of compounds to which red wine pigments (Anthocyanins) belong.<br>
 +
2 - the enzyme has been successfully expressed in <i>E. coli</i><br>
 +
3 - the enzyme activity can be tested using spectophotography
 
</p>
 
</p>
  
 
<h3>Polyphenol degrading enzymes</h3>
 
<h3>Polyphenol degrading enzymes</h3>
<p>
 
Throughout this project we tested several polyphenol enzymes on their ability to degrade wine stains. We decided to focus on polyphenol degrading enzymes because Anthocyanins, red wine pigments, belong to this group of proteins. <br>In order to choose the enzymes with which to work with, we analysed the literature and applied two different criteria: first, we chose enzymes that had been expressed in <i>E. coli</i> before, and second, enzymes whose activity could be tested using spectrography. This two criteria were chosen to make our project realistic, both given the time we had for the project and the facilities we had at our disposal. <br>First, we decided to work with the Polyphenol Oxidase from <i>Camellia sinensis</i> (<i>PPO</i>). We chose this enzyme because Anthocyanins had been already described as natural substrates for it (Jaiswal 2009). In a study where the authors expressed the enzyme in <i>E. coli</i>, a structure-activity analysis was performed, and it was determined that the optimal way of expressing this enzyme in <i>E. coli</i> was to eliminate the signal peptide region that guided the protein to cell wall in plant cells (Wu 2010). We therefore cloned a codon optimised version of the gene, without the signal peptide, called <i>PPO2</i>. <br>We decided to work with <i>Bacillus pumilus</i>’ BpuI laccase as well, because laccases have been shown to degrade Anthocyanins (Rodriguez Couto 2006). <i>B. subtilis</i>’ laccase already existed in the Biobrick registry for iGEM (BBa_K863000), so we took the already existing sequence and codon optimised it for <i>E. coli</i>. <br>
 
Next, we decided to work with two Catechol-dioxygenases, XylE from <i>Pseudonomas putida</i> and CatA from <i>Acinetobacter pittii</i>. These two enzymes have Catechol as substrate, which shares structural similarities with Anthocyanins. We thought that these enzymes would be strong candidates for removing red-wine stains, specially XylE, given that it had been described as being a promiscuous enzyme (Cerdan 1995, Kobayasi 1995 and Lin 2015). <br>The last enzyme we decided to work with was <i>Lactobacillus plantarum</i>’s tannin acyl hydrolase TanLpI. Tannins are Polyphenol molecules that are important for wine colour. Although the red colour associated with red wine comes from Anthocyanins, Tannins are important molecules that need to form covalent bonds with Anthocyanins in order for them to be stable over time. We thought that an alternative way of getting rid of wine stains would be to attack the Tannin molecules, hence making the Anthocyanin ones unstable. We chose to work with this specific tannin acyl hydrolase because it fit the criteria we exposed before (Curiel 2009). <br>
 
</p>
 
  
<h3>Design of our expression system </h3>
 
 
<p>
 
<p>
The first step of this project was to design a system that allowed us to test all our proteins in an easy way. We aimed to create a method that would allow us to use the same primers for all the versions of the proteins, as well as a system that would make it easy for then transit our proteins to the Phytobricks. <br>The first step was to obtain the codon optimised sequences of the five proteins we decided to work with. We obtained gBlocks of all the proteins, with a histidine tail on the C-terminus. We designed the synthetic fragment so that it would allow us test the proteins both with and without the histidine tag. Two primers common to all proteins were designed for inserting the protein sequences with the his-tag on our backbone. A third primer, individual to each of the proteins, was designed as well, to allow for cloning of the protein sequence alone, just in case the his-tag inhibited the protein activity. All primers incluses <i> Bsa</i>I restriction sites. <br>
+
Our literature search resulted in six enzymes that met these criteria. <br>
 +
The first enzyme of interest was the natural anthocyanin degrading<b> Polyphenol Oxidase (PPO)</b> from <i>Camellia sinensis</i> (<i>PPO</i>) (Jaiswal 2009). In order to optimally express this enzyme, we removed the naturally occurring signal peptide region of PPO and annotated the resulting enzyme as PPO2. Wu and coworkers reported that this signal peptide hampered the heterologous expression in <i>E. coli</i> (Wu <i>et al.</i> 2010). <br>
 +
We also selected the <b>Beta-galactosidase</b> from <i>Vitis bellula</i>, <b>BG1</b>. This enzyme was a strong candidate for our assays since it was shows to degrade anthocyanins, but unfortunately we did not manage to express it correctly in <i>E. coli</i>. <br>
 +
<i>Bacillus pumilus</i>’ <b>BpuI laccase</b> caught our attention, as laccases have been shown to degrade anthocyanins (Rodriguez Couto 2006). Notably, its corresponding gene was available as BioBrick BBa_K863000 in the iGEM Part Registry. <br>
 +
Next, we wanted to test two <b>Catechol-dioxygenases: XylE </b>from <i>Pseudonomas putida</i> and <b>CatA </b>from <i>Acinetobacter pittii</i>. We hypothesized that these enzymes would be strong candidates for removal of red-wine stains because catechol shares important structural similarities with anthocyanin (Cerdan 1995, Kobayasi 1995 and Lin 2015).<br>
 +
Finally, we decided to work with <i>Lactobacillus plantarum</i>’s <b>tannin acyl hydrolase TanLpI</b>. Although the red wine colour is associated with anthocyanins, tannins are important colour stabilizers. Tannins molecules form covalent bonds with anthocyanins to provide molecular stability of the anthocyanins over time. By targeting the tannin molecules, we wanted to destabilize the anthocyanins and thereby indirectly remove the wine stain (Curiel 2009).
 
</p>
 
</p>
  
 
<div id="figurebox">
 
<div id="figurebox">
<img src="https://static.igem.org/mediawiki/2016/b/b2/Paris_Bettencourt-Binding_Domain.jpeg" alt="Quercetin strains degradation" style="width:900px;">
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<img src="https://static.igem.org/mediawiki/2016/d/d7/Paris_Bettencourt-enzyme_hereitcomes.png" alt="Quercetin strains degradation" style="width:900px;">
 
<p>
 
<p>
<b> Figure X: Figure of the design</b> Explaining the image a bit
+
<b> Figure 1</b> Targets of our enzymes within the malvidin molecule. This figure shows the place in which each of the enzymes attack the malvidin molecule. The different coloured lightbolts represent the different enzymes.
 
</div>
 
</div>
  
 +
<h3>Design of our expression system</h3>
 
<p>
 
<p>
In order to have a plasmid that allowed for overexpression of our proteins, we adapted the pCOLA plasmid from the pDUET system to make it compatible with Golden Gate assembly. We deleted all <i> Bsa</i>I restriction sites and synthesised a plasmid with only one T7 promoter. We placed a <i> Bsa</i>I restriction site on the desired position so as to be able to insert our genes under the T7 promoter. <br>In the end, we had a simple workflow for expressing our proteins under the T7 promoter. We constructed all the plasmids with Golden Gate, replicated them in <i>E. coli</i> DH5alfa and transformed them in <i>E. coli</i> BL21(DE3) for protein expression.
+
We constructed recombinant <i>E. coli</i> strains carrying the selected candidate enzymes using the following strategy. First, all the genes corresponding to the enzymes described above were codon optimized for expression in <i>E. coli</i> and equipped with an histidine tag at their C-terminus to facilitate protein purification. We adopted the pCOLA plasmid from the pDUET system to overexpress each enzyme (Novagen). In order to make the pCOLA plasmid compatible with the Golden Gate assembly method, we deleted all <i>Bsa</i>I restriction sites and replaced the original multiple cloning site downstream of the T7 promoter for a LacZ cassette. Two <i>Bsa</i>I restriction sites were added to allow the insertion of any gene of interest between the T7 promoter and T7 terminator while removing LacZ. <br>
 +
This way, we had a simple and standardised workflow for the construction of our expression plasmids. Golden gate reactions were transformed to <i>E. coli</i> DH5-alpha cells and plated on X-Gal/IPTG plates to allow for blue/white screening. The verified constructs were transformed to <i>E. coli</i> BL21(DE3) to overexpress the proteins in the presence of IPTG.
 
</p>
 
</p>
  
<h3>Fabric Binding Domain fusion proteins - enhancing of wine stain degrading properties</h3>
+
<h3>Fabric Binding Domain fusion proteins</h3>
 
<p>
 
<p>
An important part of our project was to enhance the ability of the chosen enzymes to degrade wine stains. Our aim was to enhance the stain removal capacity of our enzymes by directing their activity to the fabric. We believed that by increasing the time of contact between our enzymes and the stain, we would get an enhancement on stain removal. <br>
+
An important part of our project was to specify the localization of the potential stain removing enzymes. We hypothesized that by increasing the time of contact between our enzymes and the substrate, we could more efficiently remove stains.  
We looked for ways of enhancing enzymatic activity in the literature, and one usual method we found was by creating fusion proteins. There are several examples of this methodology. For example, by fusing either a transcriptional repressor or an transcriptional activator to dCas9, enhanced repression and enhanced activation of genes can be accomplished. This happens because dCas9 guides the repressor or the activator to the desired place of action, and therefore the process is more efficient. <br>
+
In nature, peptide-based binding domains play an important role in the localization of an enzyme. Using the same principle, we fused proteins to the peptides we found to have a binding affinity for cotton, wool, silk and linen. (<a href="https://2016.igem.org/Team:Paris_Bettencourt/Project/Binding">More information on the Fabric Binding Domains.</a>) These peptides were thoroughly tested to see whether they were able to bind to specific type of fabrics. <br> To quantify the fabric binding affinity in the context of a fusion protein, we assayed their behaviour when fused to GFP. For the near future, we would like to test a larger number of enzymatic agents fused to a variety of fabric binding domains. This way, we have paved the way to expand the way stains can be treated with biological solutions.
Inspired by this examples, we wanted to find something that would guide our enzymes to the fabric in which the stains were found. We found peptides with affinity for specific fabrics using phage display. <ahref="https://2016.igem.org/Team:Paris_Bettencourt/Project/Binding">More information on the FBD.</a> These peptides were thoroughly tested to see whether they were able to bind to specific type of fabrics. <br>
+
Nonetheless we wanted to know whether the binding of these Fabric Binding Domains (FBD) would affect the activity of our proteins. We tested them first with GFP and then with some of our enzymes. Testing them with GFP allowed us to know in a straightforward way whether it was possible to express functional FBD-fusion proteins.
+
 
</p>
 
</p>
  
<h2 class="red">Results</h2>
+
 
 +
<h2 class="red" id="ancre">Results</h2>
  
 
<h3>Outline of the project</h3>
 
<h3>Outline of the project</h3>
 +
 +
<div id="figurebox">
 +
<img src="https://static.igem.org/mediawiki/2016/b/bf/Paris_Bettencourt-enzyme_flowchart.png" alt="Quercetin strains degradation" style="width:900px;">
 
<p>
 
<p>
Text about the outline
+
<b> Figure 2</b> Outline of the enzyme project. The enzyme sub-project has two main parts: the first one is the testing of a set of polyphenol degrading enzymes on their ability to degrade Anthocyanins, and the second one is the directing of that ability for stain removal purposes by fusing the enzymes to the Fabric Binding Domains (FBD) found by our team. <br>
 +
First, the different genes are codon optimised and cloned in <i>E. coli</i>. The heterologously expressed enzymes are then tested with their natural substrate to check whether they are functional of not. <br>
 +
Once their functionality is confirmed, we move on to the fusion of the enzymes with the FBD. To test whether it is feasible to fuse proteins with the FBD without disrupting their activity, we first create GFP-FBD fusion proteins for each of the FBDs and test their fluorescence. We also test the affinity of the resulting fusion proteins on different fabrics. This allows us to confirm whether it is feasible to construct fusion proteins in which both the enzyme and the FBD's activities are functional. <br>
 +
Having confirmed that fusion proteins are able to maintain both their enzymatic activity and their binding activity, we create fusion proteins with our polyphenol degrading enzymes and the FBDs. We test the activity of the resulting fusion proteins on the natural substrate of each of the original enzymes. <br>
 +
The last step is to test the original enzymes and the FBD-fusion versions on their ability to degrade Anthocyanins. We envision doing this in two ways: by testing them on Malvidin directly and by testing them on fabrics stained with wine.
 
</p>
 
</p>
 +
</div>
 +
  
 
<h3>Confirming expression of our proteins</h3>
 
<h3>Confirming expression of our proteins</h3>
 +
 +
<p>After obtaining <i>E. coli</i> BL21(DE3) strains able to express our polyphenol degrading enzymes, we decided to focus our work on four of them, BpuI, CatA, XylE and BG1. We decided to postpone the experiments with PPO2, since it forms inclusion bodies that would demands a complicated setup to make the protein functional. We also decided to postpone the experiments on TanLpI due to the fact that its measurement required stopping the reaction and coupling a second reaction to measure the product. Therefore, the experimental setup was complex as well. <br>
 +
The first step was to check whether we were correctly expressing our proteins of choice. Figure 3 shows an SDS-PAGE analysis of the cell extracts performed with the cells in which expression of the proteins was induced. The cell extract protocol can be seen in the methods part of this project.
 +
</p>
 +
 +
<div id="figurebox">
 +
<img src="https://static.igem.org/mediawiki/2016/f/f5/Paris_Bettencourt-gelprotein.png" alt="Quercetin strains degradation" style="width:900px;">
 
<p>
 
<p>
Text about the SDS-PAGE gels
+
<b>Figure 3</b> SDS-PAGE gels for expression of BpuI, CatA and XylE. Sample preparation: <i>E.coli</i> BL21(DE3) cells expressing the proteins were induced for 5 hours with 0.5mM IPTG. After 5 hours, the OD(600nm) was measured, the cells centrifuged and the pellet was resuspended in Laemmili sample buffer to a final OD of 10. The cells were cooked at 95ºC for  10 min, and 10uL of the resulting solution were loaded on the gel. Ladder used: Kaleidoskope. Staining: The gel was washed 3x with miliQ water to remove the SDS and staining was performed using BioRad Comassie for 30min. De-staining was performed by leaving the gel in miliQ water for 1 hour with gentle shaking.<br>
 +
As expected, no overexpression was observed in BL21(DE3), whether it was induced or not induced. BpuI was correctly overexpressed, with the observed size being the expected, around 59kDa. CatA overexpression was very mild, but the correct sized band was observed, of around 34kDa. XylE was correctly overexpressed, with the correct band being observed, of around 36kDa.
 +
</p></div>
 +
 
 +
<p>Overexpression of three of the four enzymes was achieved. BG1, although very promising, was not correctly expressed, and therefore we continued our experiments with BpuI, CatA and XylE alone.
 
</p>
 
</p>
  
 
<h3>Testing our enzymes in their natural substrate</h3>
 
<h3>Testing our enzymes in their natural substrate</h3>
 +
 +
<p>The next natural step was to test whether the overexpressed proteins were functional. We performed cell extracts of BpuI, CatA and XylE enzymes and measured their activity using the appropriate substrates.
 +
</p>
 +
 +
<div id="figurebox">
 +
<img src="https://static.igem.org/mediawiki/2016/d/d9/Paris_Bettencourt-enzymesagain.jpg" alt="Quercetin strains degradation" style="width:900px;">
 
<p>
 
<p>
Text about the tests with Catechol.
+
<b>Figure 4</b> Confirmation of enzymatic activity of heterologously expressed BpuI, CatA and XylE. Results obtained using the cell extracts prepared as explained on the methods section of this page. Control corresponds at BL21(DE3) background, using  cell extracts of BL21(DE3) cells that do not express our proteins. In all cases, values measured correspond to reaction product.<br>
 +
<b>A</b>. BpuI activity was measured in Citrate Phosphate Buffer at pH 4, with 0.4mM of ABTS being used as substrate, as recommended in the literature. Measurements were taken after 90min, timepoint at which the substrate present in the reaction medium was finished. <br>
 +
<b>B</b>. CatA activity was measured in Sodium Phosphate 50mM at pH 7, with 30mM of Catechol as substrate, as recommended in the literature. Measurements were taken after 35 min, timepoint at which all the substrate had been consumed. <br>
 +
<b>C</b>. XylE activity was measured in Potassium Phosphate 100mM at pH 7.5, with 30mM of Catechol as substrate, as recommended in the literature. Measurements were taken after 12 min, timepoint after which all the substrate had been consumed.
 +
</p></div>
 +
 
 +
<p>In all cases, the proteins were demonstrated to be functional. All three cell extracts carrying them were able to degrade their natural substrates more than the control.
 
</p>
 
</p>
  
 
<h3>Testing the effect of fusing FBD with proteins</h3>
 
<h3>Testing the effect of fusing FBD with proteins</h3>
<p>
+
 
Text about the tests with the GFP-FBD. Also about the binding of the FBD to our proteins!
+
<p>The following step was to construct our fusion proteins, fusing Fabric Binding Domains to our best performing enzymes.<br>
 +
First, we needed to understand whether it was feasible to build a functional FBD-enzyme fusion protein. We needed to understand if the joining together of the two parts would have a negative effect on the binding capacity or in the enzyme itself.<br>
 +
An easy way of testing the feasibility of these constructs is to test the FBDs with GFP first. We constructed fusion proteins with the several FBD on the N'-terminal, since it was the terminal the binding domains were recommended to be placed at.
 
</p>
 
</p>
  
<h3>Testing our enzymes on their ability to degrade Anthocyanins</h3>
+
<div id="figurebox">
 +
<img src="https://static.igem.org/mediawiki/2016/f/fe/Paris_Bettencourt-enzyme_heatmaps.jpg" alt="Quercetin strains degradation" style="width:800px;">
 
<p>
 
<p>
Text about the tests with Anthocyanins.
+
<b>Figure 5</b> Analysis of FBD-GFP fusion proteins. With this image we show that the GFP activity was not affected by the fusion between the FBDs and the GFP. CBD stands for the +control, Cellulose Binding Domain from the Biobrick registry BBa_K1321357. <br>
 +
Cell extract from strains expressing our fusion proteins was incubated overnight with the fabrics. Afterwards two washes of the fabrics were performed. Water, PBS, BSA 5%, Ethanol 70% and Catechol 30mM correspond to the washing solutions we used to remove the non-bound GFP, as well as to test the binding strength of the different peptides. The data displayed corresponds to the values after the final wash, normalised using the values from the first wash. <br>
 +
The intensity of the colour corresponds to the GFP signal measured at that point. <br
 +
Each heatmap corresponds to the results obtained with different fabrics. Results were obtained using the final 96-wells-based assay designed by our Assay team, using untreated, unbleached cotton, linen, silk and wool.  
 
</p>
 
</p>
 +
</div>
  
<h3>Testing our enzymes on their ability to degrade wine stains</h3>
+
<p>In figure 5 we can see that some FBDs show high affinity to certain fabrics, and some are even specific. In the case of FBD2, it shows high affinity and specificity for both cotton and linen. This FBD binds poorly to silk and wool, which is interesting given that both cotton and linen are plant-fiber-based, and silk and wool come from animal sources. <br>
 +
FBD5 shows affinity to all four fabrics, and has no specificity for any of them.<br>
 +
Interestingly, most FBDs have affinity for silk, which is very useful in the development of our product given that through our human practises we were able to get to know that silk is one of the most demanding fabrics when it comes to stain removal.<br>
 +
Different washing solutions also influenced differently the several FBDs. For example, FB10 performs very well when washed with water in silk and wool, but performs very badly when washed with the same solution in cotton and linen. <br><br>
 +
<pHaving checked that the creation of these fusion proteins was possible, we proceeded to create four fusion proteins. We fused FBD1 (binds to all fabrics) and FBD10 (binds to cellulose) to CatA and XylE.
 +
</p>
 +
 
 +
<div id="figurebox">
 +
<img src="https://static.igem.org/mediawiki/2016/a/a8/Paris_Bettencourt-enzyme_thisonepleasework7.jpg" alt="Quercetin strains degradation" style="width:450px; float:left;">
 
<p>
 
<p>
Text about the tests with wine on fabric.
+
<b>Figure 6</b> Confirmation of enzymatic activity of the fusion proteins CatA-FBD1, CatA-FBD10, XylE-FBD1 and XylE-FBD10. Results obtained using the cell extracts prepared as explained on the methods section of this page. Control corresponds at BL21(DE3) background, using  cell extracts of BL21(DE3) cells that do not express our proteins. In all cases, values measured correspond to reaction product.<br>
 +
<b>In green</b>. CatA fusion proteins' activity was measured in Sodium Phosphate 50mM at pH 7, with 30mM of Catechol as substrate, as recommended in the literature. Measurements were taken after 35 min, timepoint at which all the substrate had been consumed by the native protein. <br>
 +
<b>In red</b>. XylE fusion proteins' activity was measured in Potassium Phosphate 100mM at pH 7.5, with 30mM of Catechol as substrate, as recommended in the literature. Measurements were taken after 12 min, timepoint after which all the substrate had been consumed by the native protein.
 +
</p> </div>
 +
 
 +
<p> Figure 6 can have two interpretations: either the binding of the FBDs affects the activity of the proteins, or it lowers their expression. Both interpretations would explained the decrease in the activity that we observed. Nonetheless, the enzymes were still active, except XylE-FBD1.
 
</p>
 
</p>
  
 +
<h3>Testing our enzymes on their ability to degrade Anthocyanins</h3>
 +
 +
<p>The next step is to test our enzymes fused with the FBDs on their ability to degrade anthocyanins. A similar assay to the one performed with Catechol will be performed using Malvidin or a solution of Anthocyanins.
 +
</p>
 +
 +
<h3>Testing our enzymes on their ability to degrade wine stains</h3>
 +
 +
<p>Following the assay with the Anthocyanins, a similar assay to that performed with the fabric assays on the FBD-CBD fusion proteins will be performed, only this time it will be carried out with fabrics stained with wine, on the assay developed by the assay team and analysed with the software designed by the same team as well.
 +
</p>
  
 
<h2 class="red">Methods</h2>
 
<h2 class="red">Methods</h2>
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<h3>Codon optimisation of plant and bacterial genes of interest</h3>                           
 
<h3>Codon optimisation of plant and bacterial genes of interest</h3>                           
 
<p>
 
<p>
One important step when expressing heterologously genes in <i>E. coli</i>, specially those of eukaryotic origin, is to perform Codon Optimisation. Different organisms have different codon preferences, which means that each organism has different abundances of the several codons that translate to a same amino acid. <br>
+
Because every organism has different codon preferences, Genes of enzyme candidates were codon optimized for expression of  in E. coli using  using the IDT Codon Optimisation tool.
We performed Codon Optimisation using the <ahref="https://eu.idtdna.com/CodonOpt">IDT Codon Optimisation tool.</a>
+
 
</p>
 
</p>
  
 
<h3>Golden Gate and Gibson Assembly for Biobrick Construction</h3>                           
 
<h3>Golden Gate and Gibson Assembly for Biobrick Construction</h3>                           
 
<p>
 
<p>
We built our plasmids using either Golden Gate or Gibson Assembly. <br>
+
We used  Golden Gater and Gibson Assembly as cloning methods to construct our plasmid
In the case of <b>Golden Gate</b>, we used NEB’s Golden Gate Assembly mix. 40fmol of backbone and 40fmol of insert were mixed in a 10uL total reaction, following the instructions provided by NEB. The mixure was incubated with the following cycle: 37ºC for 5 min, (37ºC for 2 min, 16ºC for 5min)x50, 37ºC for 5min, 50ºC for 10min and 80ºC for 10min. <br>
+
In our <b>Golden Gate</b> reactions, we used NEB’s Golden Gate Assembly mix,. 40 fmol of backbone and 40 fmol of insert following the instructions provided by NEB. The mixture was incubated with the following cycle: 37ºC for 5 min, (37ºC for 2 min, 16ºC for 5min)x50, 37ºC for 5min, 50ºC for 10min and 80ºC for 10min. <br>
In the case of <b>Gibson Assembly</b>, 200ng of DNA (backbone and insert with a ratio of 1:2) were mixed in a 15uL total reaction, using NEB’s 2X Hifi DNA Assembly MasterMix. The mixure was incubated 1h at 37ºC.  
+
When we performed <b>Gibson Assembly</b> reactions, 200ng of DNA (backbone and insert with a ratio of 1:2) were mixed in a 15uL total reaction, using NEB’s 2X Hifi DNA Assembly MasterMix. The mixure was incubated 1h at 37ºC.<br>
 
</p>
 
</p>
  
 
<h3>Induced protein expression by the use of expression systems</h3>                           
 
<h3>Induced protein expression by the use of expression systems</h3>                           
 
<p>
 
<p>
We used <i>E. coli</i> BL21(DE3) as our expression strain. Cells transformed with our constructs were incubated at appropriate temperature until they reached an OD(600nm) of 0.4-0.6. They were then induced with 0.5mM IPTG.
+
We used <i>E. coli</i> BL21(DE3) as our expression strain. Cells transformed with our constructs were incubated at appropriate temperature until they reached an OD(600nm) of 0.4-0.6. They were then induced with 0.5mM IPTG for 5 hours.
 
</p>
 
</p>
  
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<h3>SDS-PAGE analysis</h3>                           
 
<h3>SDS-PAGE analysis</h3>                           
 
<p>
 
<p>
We analysed our proteins using pre-made, 10% SDS-PAGE gels from BioRad.
+
We analysed our proteins using pre-made, 10% SDS-PAGE gels from BioRad. As a ladder we used Kaledoskope from BioRad.
 
</p>
 
</p>
  
<h3>Assaying the binding strength of the Fabric Binding Domains <ahref="https://2016.igem.org/Team:Paris_Bettencourt/Project/Binding">More information on the FBD</a></h3>                           
+
<h3>Assaying the binding strength of the Fabric Binding Domains.</h3>                           
 
<p>
 
<p>
We assayed the strength or the Fabric Binding Domains using the <ahref="https://2016.igem.org/Team:Paris_Bettencourt/Project/Assay">assay</a> developped by our team, carrying cotton, wool, silk and linnen untreated, unbleached fabrics. Cell extracts of BL21(DE3) strains carrying GFP-FBD fusion proteins were performed and 1:10 dilutions were made. The diluted cell extracts were incubated in the 96-well-plates-based assay with the fabrics overnight. The several measurements were performed in a Infinite M200-Pro Tecan plate reader.  
+
We assayed the strength or the Fabric Binding Domains using the assay developed by our team, carrying cotton, wool, silk and linen untreated, unbleached fabrics. Cell extracts of BL21(DE3) strains carrying GFP-FBD fusion proteins were performed and 1/10 dilutions were made. The diluted cell extracts were incubated in the 96-well-plates-based assay with the fabrics overnight. Several washes of the fabrics were performed using water, PBS, BSA 5%, Ethanol 70% and Catechol 0.03M. The several measurements were performed in a Infinite M200-Pro Tecan plate reader.  
 
</p>
 
</p>
  
 
<h3>Assaying the enhanced stain removal activity of fusion proteins</h3>                           
 
<h3>Assaying the enhanced stain removal activity of fusion proteins</h3>                           
 
<p>
 
<p>
We assayed the stain removal activity of our fusion proteins in our assay, with the different fabrics stained with wine. The different measurements wera carried out in the Infinite M200-Pro Tecan plate reader.  
+
We assayed the stain removal activity of our fusion proteins in our assay, with the different fabrics stained with wine. The different measurements wera carried out in the Infinite M200-Pro Tecan plate reader.<br>
 +
BpuI activity was measured in Citrate Phosphate Buffer at pH 4, with 0.4mM of ABTS being used as substrate, as recommended in the literature. Measurements were taken at 420nm. <br>
 +
CatA activity was measured in Sodium Phosphate 50mM at pH 7, with 30mM of Catechol as substrate, as recommended in the literature. Measurements were taken at 260nm.<br>
 +
XylE activity was measured in Potassium Phosphate 100mM at pH 7.5, with 30mM of Catechol as substrate, as recommended in the literature. Measurements were taken at 475nm.
 
</p>
 
</p>
 
 
 
<h2 class="red">Attributions</h3>
 
<h2 class="red">Attributions</h3>
 
<p>
 
<p>
This project was done by Alicia and Sebastián. BpuI data was obtained by Mislav. A special thanks to our advisor Nadine for all the insight on Golden Gate and for the GFP sequences. A special thanks to our advisor Jake for all the help with the figures and the pDUET system. Thank you to Afonso Bravo for the help with the his-tag purification. Thank you to IDT for the synthesis of our genes, and thanks to NEB for the enzymatic kits.
+
This project was done by Alicia Calvo-Villamañán and Sebastián Sosa-Carrillo. BpuI data was obtained by Mislav Acman. A special thanks to our advisor Nadine Bongaerts for all the insight on Golden Gate and for the GFP sequences. A special thanks to our advisor Jake Wintermute for all the help with the figures and the pDUET system. Thank you to Afonso Bravo for the help with the his-tag purification. Thank you to IDT for the synthesis of our genes, and thanks to NEB for the enzymatic kits.
 
</p>
 
</p>
 
+
<img src="https://static.igem.org/mediawiki/2016/f/fb/Paris_Bettencourt-sebasstatic.jpeg"/><img src="https://static.igem.org/mediawiki/2016/9/95/Paris_Bettencourt-Aliciastatic.jpeg"/>
 
<h2 class="red">References</h2>
 
<h2 class="red">References</h2>
 
<ul>
 
<ul>
 +
 +
<li>Barbagallo, R.N., Palmeri, R. Fabiano, S.; Rapisarda, P.; ,Spagna, G (2007). Characteristic of beta-glucosidase from Sicilian blood oranges in relation to anthocyanin degradation.Enzymatic Microbial Technology. 41, 570-575
 
<li>Cerdan P, Rekik M and Harayama S (1995). Substrate specificity differences between two catechol 2,3-dioxygenases encoded by the TOL and NAH plasmids from <i>Pseudomonas putida</i>. European Journal of Biochemistry. 229, 113-118
 
<li>Cerdan P, Rekik M and Harayama S (1995). Substrate specificity differences between two catechol 2,3-dioxygenases encoded by the TOL and NAH plasmids from <i>Pseudomonas putida</i>. European Journal of Biochemistry. 229, 113-118
  
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Latest revision as of 03:27, 20 October 2016




Goals

  • Identification of potential Anthocyanin degrading enzymes.
  • Heterologous expression of the potential Anthocyanin degrading enzymes in E. coli.
  • Enhancement of the enzymatic wine stain degradation through the construction of potential Anthocyanin degrading enzymes fused to Fabric Binding Domains. More information on FBD on Binding.

BioBricks

  • BBa_K2043001
  • BBa_K2043002
  • BBa_K2043003
  • BBa_K2043004
  • BBa_K2043005
  • BBa_K2043006
  • BBa_K2043007
  • BBa_K2043008
  • BBa_K2043009
  • BBa_K2043010
  • BBa_K2043011
  • BBa_K2043012
  • BBa_K2043013
  • BBa_K2043015
  • BBa_K2043016
  • BBa_K2043017

Results

  • Six candidate plant and bacterial enzymes were tested for expression in E. coli
  • Three out of six enzymes were functionally expressed in E. coli and tested for their ability to degrade their natural substrate
  • Finally, the several enzymes were fused with Fabric Binding Domains and their activity was still observed. FBD-GFP fusion proteins revealed affinity and specificity in our FBDs

Methods

  • Codon optimisation of plant and bacterial genes of interest
  • Golden Gate and Gibson Assembly for Biobrick Construction
  • Transformation of E. coli cells
  • Induced protein expression by the use of expression systems
  • Preparation of cell extract for protein activity testing
  • Assaying the binding strength of the Fabric Binding Domains. More information on the FBD on Binding
  • Assaying the enhanced stain removal activity of fusion proteins

Abstract

Anthocyanins, the key pigments present in wine, are polyphenolic molecules that are naturally found in many plants. In this part of our iGEM project we turn to nature in the search for enzymes with the potential ability to degrade these pigments for expression in E. coli. Three potential wine stain removers, BpuI, CatA and XylE, were successfully expressed in E. coli and are ready to be tested for their ability to degrade Anthocyanins. To take their stain removal capacity to the next level, we discovered peptide binding domains that supported the localization of the enzymes to cotton, wool, linen and silk. Here we also showed that enzymes XylE and CatA maintained their activity when fused to our Fabric Binding Domains. We believe that our fabric-specific enzyme detergents have opened a new door for the development of innovative laundry products.

Motivation and Background

Throughout human history, people have taken inspiration from nature to find solutions for everyday problems. A famous example is velcro. Velcro was inspired by the way the structure of plant burrs allowed them to attach to the furry bodies of animals.
As technology has progressed, we not only take inspiration from nature, but also control and enhance biology for our specific purposes.
We adopted this mindset when we approached this part of our project: the development of anthocyanin degrading enzymes that can be attached to a specific type of fabric.
First, we searched for enzyme candidates that could be suitable within this scope. We selected enzymes from literature based on the following three criteria:
1- the enzyme has the known ability to specifically degrade Anthocyanins, or, more broadly, polyphenols, which are the class of compounds to which red wine pigments (Anthocyanins) belong.
2 - the enzyme has been successfully expressed in E. coli
3 - the enzyme activity can be tested using spectophotography

Polyphenol degrading enzymes

Our literature search resulted in six enzymes that met these criteria.
The first enzyme of interest was the natural anthocyanin degrading Polyphenol Oxidase (PPO) from Camellia sinensis (PPO) (Jaiswal 2009). In order to optimally express this enzyme, we removed the naturally occurring signal peptide region of PPO and annotated the resulting enzyme as PPO2. Wu and coworkers reported that this signal peptide hampered the heterologous expression in E. coli (Wu et al. 2010).
We also selected the Beta-galactosidase from Vitis bellula, BG1. This enzyme was a strong candidate for our assays since it was shows to degrade anthocyanins, but unfortunately we did not manage to express it correctly in E. coli.
Bacillus pumilusBpuI laccase caught our attention, as laccases have been shown to degrade anthocyanins (Rodriguez Couto 2006). Notably, its corresponding gene was available as BioBrick BBa_K863000 in the iGEM Part Registry.
Next, we wanted to test two Catechol-dioxygenases: XylE from Pseudonomas putida and CatA from Acinetobacter pittii. We hypothesized that these enzymes would be strong candidates for removal of red-wine stains because catechol shares important structural similarities with anthocyanin (Cerdan 1995, Kobayasi 1995 and Lin 2015).
Finally, we decided to work with Lactobacillus plantarum’s tannin acyl hydrolase TanLpI. Although the red wine colour is associated with anthocyanins, tannins are important colour stabilizers. Tannins molecules form covalent bonds with anthocyanins to provide molecular stability of the anthocyanins over time. By targeting the tannin molecules, we wanted to destabilize the anthocyanins and thereby indirectly remove the wine stain (Curiel 2009).

Quercetin strains degradation

Figure 1 Targets of our enzymes within the malvidin molecule. This figure shows the place in which each of the enzymes attack the malvidin molecule. The different coloured lightbolts represent the different enzymes.

Design of our expression system

We constructed recombinant E. coli strains carrying the selected candidate enzymes using the following strategy. First, all the genes corresponding to the enzymes described above were codon optimized for expression in E. coli and equipped with an histidine tag at their C-terminus to facilitate protein purification. We adopted the pCOLA plasmid from the pDUET system to overexpress each enzyme (Novagen). In order to make the pCOLA plasmid compatible with the Golden Gate assembly method, we deleted all BsaI restriction sites and replaced the original multiple cloning site downstream of the T7 promoter for a LacZ cassette. Two BsaI restriction sites were added to allow the insertion of any gene of interest between the T7 promoter and T7 terminator while removing LacZ.
This way, we had a simple and standardised workflow for the construction of our expression plasmids. Golden gate reactions were transformed to E. coli DH5-alpha cells and plated on X-Gal/IPTG plates to allow for blue/white screening. The verified constructs were transformed to E. coli BL21(DE3) to overexpress the proteins in the presence of IPTG.

Fabric Binding Domain fusion proteins

An important part of our project was to specify the localization of the potential stain removing enzymes. We hypothesized that by increasing the time of contact between our enzymes and the substrate, we could more efficiently remove stains. In nature, peptide-based binding domains play an important role in the localization of an enzyme. Using the same principle, we fused proteins to the peptides we found to have a binding affinity for cotton, wool, silk and linen. (More information on the Fabric Binding Domains.) These peptides were thoroughly tested to see whether they were able to bind to specific type of fabrics.
To quantify the fabric binding affinity in the context of a fusion protein, we assayed their behaviour when fused to GFP. For the near future, we would like to test a larger number of enzymatic agents fused to a variety of fabric binding domains. This way, we have paved the way to expand the way stains can be treated with biological solutions.

Results

Outline of the project

Quercetin strains degradation

Figure 2 Outline of the enzyme project. The enzyme sub-project has two main parts: the first one is the testing of a set of polyphenol degrading enzymes on their ability to degrade Anthocyanins, and the second one is the directing of that ability for stain removal purposes by fusing the enzymes to the Fabric Binding Domains (FBD) found by our team.
First, the different genes are codon optimised and cloned in E. coli. The heterologously expressed enzymes are then tested with their natural substrate to check whether they are functional of not.
Once their functionality is confirmed, we move on to the fusion of the enzymes with the FBD. To test whether it is feasible to fuse proteins with the FBD without disrupting their activity, we first create GFP-FBD fusion proteins for each of the FBDs and test their fluorescence. We also test the affinity of the resulting fusion proteins on different fabrics. This allows us to confirm whether it is feasible to construct fusion proteins in which both the enzyme and the FBD's activities are functional.
Having confirmed that fusion proteins are able to maintain both their enzymatic activity and their binding activity, we create fusion proteins with our polyphenol degrading enzymes and the FBDs. We test the activity of the resulting fusion proteins on the natural substrate of each of the original enzymes.
The last step is to test the original enzymes and the FBD-fusion versions on their ability to degrade Anthocyanins. We envision doing this in two ways: by testing them on Malvidin directly and by testing them on fabrics stained with wine.

Confirming expression of our proteins

After obtaining E. coli BL21(DE3) strains able to express our polyphenol degrading enzymes, we decided to focus our work on four of them, BpuI, CatA, XylE and BG1. We decided to postpone the experiments with PPO2, since it forms inclusion bodies that would demands a complicated setup to make the protein functional. We also decided to postpone the experiments on TanLpI due to the fact that its measurement required stopping the reaction and coupling a second reaction to measure the product. Therefore, the experimental setup was complex as well.
The first step was to check whether we were correctly expressing our proteins of choice. Figure 3 shows an SDS-PAGE analysis of the cell extracts performed with the cells in which expression of the proteins was induced. The cell extract protocol can be seen in the methods part of this project.

Quercetin strains degradation

Figure 3 SDS-PAGE gels for expression of BpuI, CatA and XylE. Sample preparation: E.coli BL21(DE3) cells expressing the proteins were induced for 5 hours with 0.5mM IPTG. After 5 hours, the OD(600nm) was measured, the cells centrifuged and the pellet was resuspended in Laemmili sample buffer to a final OD of 10. The cells were cooked at 95ºC for 10 min, and 10uL of the resulting solution were loaded on the gel. Ladder used: Kaleidoskope. Staining: The gel was washed 3x with miliQ water to remove the SDS and staining was performed using BioRad Comassie for 30min. De-staining was performed by leaving the gel in miliQ water for 1 hour with gentle shaking.
As expected, no overexpression was observed in BL21(DE3), whether it was induced or not induced. BpuI was correctly overexpressed, with the observed size being the expected, around 59kDa. CatA overexpression was very mild, but the correct sized band was observed, of around 34kDa. XylE was correctly overexpressed, with the correct band being observed, of around 36kDa.

Overexpression of three of the four enzymes was achieved. BG1, although very promising, was not correctly expressed, and therefore we continued our experiments with BpuI, CatA and XylE alone.

Testing our enzymes in their natural substrate

The next natural step was to test whether the overexpressed proteins were functional. We performed cell extracts of BpuI, CatA and XylE enzymes and measured their activity using the appropriate substrates.

Quercetin strains degradation

Figure 4 Confirmation of enzymatic activity of heterologously expressed BpuI, CatA and XylE. Results obtained using the cell extracts prepared as explained on the methods section of this page. Control corresponds at BL21(DE3) background, using cell extracts of BL21(DE3) cells that do not express our proteins. In all cases, values measured correspond to reaction product.
A. BpuI activity was measured in Citrate Phosphate Buffer at pH 4, with 0.4mM of ABTS being used as substrate, as recommended in the literature. Measurements were taken after 90min, timepoint at which the substrate present in the reaction medium was finished.
B. CatA activity was measured in Sodium Phosphate 50mM at pH 7, with 30mM of Catechol as substrate, as recommended in the literature. Measurements were taken after 35 min, timepoint at which all the substrate had been consumed.
C. XylE activity was measured in Potassium Phosphate 100mM at pH 7.5, with 30mM of Catechol as substrate, as recommended in the literature. Measurements were taken after 12 min, timepoint after which all the substrate had been consumed.

In all cases, the proteins were demonstrated to be functional. All three cell extracts carrying them were able to degrade their natural substrates more than the control.

Testing the effect of fusing FBD with proteins

The following step was to construct our fusion proteins, fusing Fabric Binding Domains to our best performing enzymes.
First, we needed to understand whether it was feasible to build a functional FBD-enzyme fusion protein. We needed to understand if the joining together of the two parts would have a negative effect on the binding capacity or in the enzyme itself.
An easy way of testing the feasibility of these constructs is to test the FBDs with GFP first. We constructed fusion proteins with the several FBD on the N'-terminal, since it was the terminal the binding domains were recommended to be placed at.

Quercetin strains degradation

Figure 5 Analysis of FBD-GFP fusion proteins. With this image we show that the GFP activity was not affected by the fusion between the FBDs and the GFP. CBD stands for the +control, Cellulose Binding Domain from the Biobrick registry BBa_K1321357.
Cell extract from strains expressing our fusion proteins was incubated overnight with the fabrics. Afterwards two washes of the fabrics were performed. Water, PBS, BSA 5%, Ethanol 70% and Catechol 30mM correspond to the washing solutions we used to remove the non-bound GFP, as well as to test the binding strength of the different peptides. The data displayed corresponds to the values after the final wash, normalised using the values from the first wash.
The intensity of the colour corresponds to the GFP signal measured at that point.

In figure 5 we can see that some FBDs show high affinity to certain fabrics, and some are even specific. In the case of FBD2, it shows high affinity and specificity for both cotton and linen. This FBD binds poorly to silk and wool, which is interesting given that both cotton and linen are plant-fiber-based, and silk and wool come from animal sources.
FBD5 shows affinity to all four fabrics, and has no specificity for any of them.
Interestingly, most FBDs have affinity for silk, which is very useful in the development of our product given that through our human practises we were able to get to know that silk is one of the most demanding fabrics when it comes to stain removal.
Different washing solutions also influenced differently the several FBDs. For example, FB10 performs very well when washed with water in silk and wool, but performs very badly when washed with the same solution in cotton and linen.

Quercetin strains degradation

Figure 6 Confirmation of enzymatic activity of the fusion proteins CatA-FBD1, CatA-FBD10, XylE-FBD1 and XylE-FBD10. Results obtained using the cell extracts prepared as explained on the methods section of this page. Control corresponds at BL21(DE3) background, using cell extracts of BL21(DE3) cells that do not express our proteins. In all cases, values measured correspond to reaction product.
In green. CatA fusion proteins' activity was measured in Sodium Phosphate 50mM at pH 7, with 30mM of Catechol as substrate, as recommended in the literature. Measurements were taken after 35 min, timepoint at which all the substrate had been consumed by the native protein.
In red. XylE fusion proteins' activity was measured in Potassium Phosphate 100mM at pH 7.5, with 30mM of Catechol as substrate, as recommended in the literature. Measurements were taken after 12 min, timepoint after which all the substrate had been consumed by the native protein.

Figure 6 can have two interpretations: either the binding of the FBDs affects the activity of the proteins, or it lowers their expression. Both interpretations would explained the decrease in the activity that we observed. Nonetheless, the enzymes were still active, except XylE-FBD1.

Testing our enzymes on their ability to degrade Anthocyanins

The next step is to test our enzymes fused with the FBDs on their ability to degrade anthocyanins. A similar assay to the one performed with Catechol will be performed using Malvidin or a solution of Anthocyanins.

Testing our enzymes on their ability to degrade wine stains

Following the assay with the Anthocyanins, a similar assay to that performed with the fabric assays on the FBD-CBD fusion proteins will be performed, only this time it will be carried out with fabrics stained with wine, on the assay developed by the assay team and analysed with the software designed by the same team as well.

Methods

Codon optimisation of plant and bacterial genes of interest

Because every organism has different codon preferences, Genes of enzyme candidates were codon optimized for expression of in E. coli using using the IDT Codon Optimisation tool.

Golden Gate and Gibson Assembly for Biobrick Construction

We used Golden Gater and Gibson Assembly as cloning methods to construct our plasmid In our Golden Gate reactions, we used NEB’s Golden Gate Assembly mix,. 40 fmol of backbone and 40 fmol of insert following the instructions provided by NEB. The mixture was incubated with the following cycle: 37ºC for 5 min, (37ºC for 2 min, 16ºC for 5min)x50, 37ºC for 5min, 50ºC for 10min and 80ºC for 10min.
When we performed Gibson Assembly reactions, 200ng of DNA (backbone and insert with a ratio of 1:2) were mixed in a 15uL total reaction, using NEB’s 2X Hifi DNA Assembly MasterMix. The mixure was incubated 1h at 37ºC.

Induced protein expression by the use of expression systems

We used E. coli BL21(DE3) as our expression strain. Cells transformed with our constructs were incubated at appropriate temperature until they reached an OD(600nm) of 0.4-0.6. They were then induced with 0.5mM IPTG for 5 hours.

Preparation of cell extract for protein activity testing

We performed our cell extract preparation using ThermoScientific’s B-PER Bacterial Protein Extraction Reagent with Enzymes.

His-tag purification of proteins

We performed the purification of our proteins using ThermoScientific’s His-Pur Ni-NTA Resin and all buffers as indicated by the manufacturer.

SDS-PAGE analysis

We analysed our proteins using pre-made, 10% SDS-PAGE gels from BioRad. As a ladder we used Kaledoskope from BioRad.

Assaying the binding strength of the Fabric Binding Domains.

We assayed the strength or the Fabric Binding Domains using the assay developed by our team, carrying cotton, wool, silk and linen untreated, unbleached fabrics. Cell extracts of BL21(DE3) strains carrying GFP-FBD fusion proteins were performed and 1/10 dilutions were made. The diluted cell extracts were incubated in the 96-well-plates-based assay with the fabrics overnight. Several washes of the fabrics were performed using water, PBS, BSA 5%, Ethanol 70% and Catechol 0.03M. The several measurements were performed in a Infinite M200-Pro Tecan plate reader.

Assaying the enhanced stain removal activity of fusion proteins

We assayed the stain removal activity of our fusion proteins in our assay, with the different fabrics stained with wine. The different measurements wera carried out in the Infinite M200-Pro Tecan plate reader.
BpuI activity was measured in Citrate Phosphate Buffer at pH 4, with 0.4mM of ABTS being used as substrate, as recommended in the literature. Measurements were taken at 420nm.
CatA activity was measured in Sodium Phosphate 50mM at pH 7, with 30mM of Catechol as substrate, as recommended in the literature. Measurements were taken at 260nm.
XylE activity was measured in Potassium Phosphate 100mM at pH 7.5, with 30mM of Catechol as substrate, as recommended in the literature. Measurements were taken at 475nm.

Attributions

This project was done by Alicia Calvo-Villamañán and Sebastián Sosa-Carrillo. BpuI data was obtained by Mislav Acman. A special thanks to our advisor Nadine Bongaerts for all the insight on Golden Gate and for the GFP sequences. A special thanks to our advisor Jake Wintermute for all the help with the figures and the pDUET system. Thank you to Afonso Bravo for the help with the his-tag purification. Thank you to IDT for the synthesis of our genes, and thanks to NEB for the enzymatic kits.

References

  • Barbagallo, R.N., Palmeri, R. Fabiano, S.; Rapisarda, P.; ,Spagna, G (2007). Characteristic of beta-glucosidase from Sicilian blood oranges in relation to anthocyanin degradation.Enzymatic Microbial Technology. 41, 570-575
  • Cerdan P, Rekik M and Harayama S (1995). Substrate specificity differences between two catechol 2,3-dioxygenases encoded by the TOL and NAH plasmids from Pseudomonas putida. European Journal of Biochemistry. 229, 113-118
  • Curiel JA, Rodriguez H, Acebron I, Mancheño JM, de las Rivas B and Muñoz R (2009). Production and physicochemical properties of recombinant Lactobacillus plantarum tannase. Journal of Agricultural Food Chemistry. 57(14), 6224-6230
  • Jaiswal V, DerManderosian A, and Porter JR. (2009). Anthocyanins and polyphenol oxidase from dried arils of pomegranate (Punica granatum L.). Food chemistry. 118(1), 11-16
  • Kobayashi T, Ishida T, Horiike K, Takara Y, Numao N, Nakazawa A, Nakazawa T and Nozaki M (1995). Overexpression of Pseudomonas putida Catechol 2,3-Dioxygenase with High Specific Activity by Genetically Engineered Escherichia coli. Journal of Biochemistry. 117, 614-622
  • Lin J and Milase RN (2015) Purification and characterization of catechol 1,2-dioxygenase from Acinetobacter sp. Y64 Strain and Escherichia coli transformants. Protein Journal. 34(6), 421-433
  • Rodriguez Couto S, Toca Herrera JL, (2006). Industrial and biotechnological applications of laccases: a review. Biotechnology Advances. 24/5), 500-513
  • Wu YL, Pan LP, Yu SL and Li HH (2010). Cloning, microbial expression and structure-activity relationship of polyphenol oxidases from Camellia sinensis. Journal of Biotechnology. 145(1), 66-72
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