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<a href="https://2016.igem.org/Team:British_Columbia">Home</a> / <a href="">Project</a> / <a href="https://2016.igem.org/Team:British_Columbia/Project/S-Layer/Cellulases">Cellulase S-Layer Engineering</a></strong>
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<a href="https://2016.igem.org/Team:British_Columbia">Home</a> /
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<a href="https://2016.igem.org/Team:British_Columbia/Project/S-Layer/Cellulases">Project - Cellulase S-Layer Engineering</a></strong>
 
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                 <section id="Abstract" class="anchor">
 
                 <section id="Abstract" class="anchor">
 
                     <h2>Abstract</h2>
 
                     <h2>Abstract</h2>
                     <p>Lignocellulosic biomass, the most abundant renewable resources in nature, represents a promising alternative to fossil fuels for sustainable production of chemicals and material. Plenty of the available lignocellulosic biomass is not a food source for humans and its industrial exploitation does not conflict with agricultural usage. However, the main technological obstacle for industrial exploitation of biomass is the recalcitrant nature of lignocellulosic polymers. One approach to harness the energy contained in biomass is to convert the lignocellulosic polymers in simple sugars, which can be further transformed in valuable compounds, using microorganisms. Some anaerobic microorganisms have developed approaches to break down recalcitrant plant biomass using elaborate extracellular enzyme complexes, containing a scaffolding protein with many attached cellulolytic enzymes. The systems were adopted by scientists to generate artificial cellulosomes. But the assembly of such complexes requires engineering of highly specific cohesin-dockerin interactions. </p>
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                     <p>Lignocellulosic biomass, the most abundantly available renewable resource in nature, represents a promising alternative to fossil fuels for sustainable production of chemicals, materials, and biofuels. The main technological obstacle for industrial exploitation of biomass is the recalcitrant nature of lignocellulosic polymers. One approach to harness the energy contained in biomass is to convert the lignocellulosic polymers into simple sugars, which can be further transformed in valuable compounds. Some anaerobic microorganisms have developed approaches to break down recalcitrant plant biomass using elaborate extracellular enzyme complexes, called cellulosomes, containing a scaffolding protein with many attached cellulolytic enzymes. The systems were adopted by scientists to generate artificial cellulosomes for biotechnological applications. These approaches have had their limitations due to inefficiencies with product yield, which  are inevitably incurred as a consequence of the metabolic strain experienced by single microbial strains that comprise most modern bioprocessing systems. </p>
                       <p> Our approach focuses on adapting the surface layer of <i>Caulobacter crescentus</i> for the highly efficient display of cellulolytic enzymes. We specifically chose <i>C. crescentus</i> as it natively expresses a two dimensional crystal lattice protein called a surface layer (S-Layer), which can be engineered to work as an enzyme display. Our goals were to embed cellulolytic enzymes into the S-Layer protein to confirm their proper folding and activity. We believe that our approach will simplify the process of "cellulosome" engineering by exploiting the robust secretion and display system of <i>Caulobacter</i>. The developed platform can be easily adapted for the display of different enzymatic activities.
+
                       <p> Our approach focuses on adapting the surface layer of <i>Caulobacter crescentus</i> for the high density display of cellulolytic enzymes. We specifically chose <i>C. crescentus</i> as it natively expresses a two dimensional crystal lattice protein called RsaA, which makes up the surface layer (S-Layer). The S-layer can be engineered for surface display of recombinant proteins. Our goal was to clone cellulolytic enzymes into the S-Layer protein to create a fusion protein for surface display, granting <i>C. crescentus</i> the ability to degrade cellulose to simple sugars. We successfully cloned, confirmed surface expression, and activity of the cellulases. Additionally, we demonstrated that surface expression of cellulases allowed <i>C. crescentus</i> to grow on cellulose as a sole carbon source, compared to wild-type <i>C. crescentus</i>, which is unable to do so. We believe that our approach will simplify the process of "cellulosome" engineering by exploiting the robust surface display system of <i>C. crescentus</i>. The developed platform can be easily adapted for the display of different proteins with different enzymatic activities.
 
                     </p>
 
                     </p>
 
                 </section>
 
                 </section>
  
 
                 <section id="Key-Achievements"
 
                 <section id="Key-Achievements"
  style="font-size: 1.15em" class="anchor">
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  style="font-size: 16px" class="anchor">
 
                     <h2>Key Achievements</h2>
 
                     <h2>Key Achievements</h2>
                         <p><li>Cloned 5 different cellulases, such as β-1,4-endoglucanase (Endo5A), β-1,4-glucosidase (Gluc1C), β-1,4-exoglucanase (CEX) and 2 versions of  β-1,4-endoglucanase(E1) in p4A723 plasmid containing rsaA protein and transformed into <i>C. crescentus</i> for the display on cell surface.</li>
+
                         <p><li>Cloned 5 different cellulases, such as β-1,4-endoglucanase (Endo5A), β-1,4-glucosidase (Gluc1C), β-1,4-exoglucanase (CEX) and 2 versions of  β-1,4-endoglucanase(E1) in p4A723 plasmid containing <i>rsaA</i> and transformed into <i>C. crescentus</i> for the display on cell surface.</li>
                         <li>Cloned 4 different cellulases - Endo5A, Gluc1C, E1, G12 into pSB1C3 and submitted as parts BBa…… </li>
+
                         <li>Cloned 4 different cellulases - Endo5A, Gluc1C, E1, G12 into pSB1C3 and submitted as parts <a href= "http://parts.igem.org/wiki/index.php?title=Part:BBa_K2139001"> BBa_K2139001</a>,<a href= "http://parts.igem.org/wiki/index.php?title=Part:BBa_K2139002"> BBa_K2139002</a>, <a href= "http://parts.igem.org/wiki/index.php?title=Part:BBa_K2139003"> BBa_K2139003, <a href= "http://parts.igem.org/wiki/index.php?title=Part:BBa_K2139004"> BBa_K2139004</a>.</li>
                         <li>Confirmed surface protein (rsaA)-cellulase fusion proteins expression of Endo5A, Gluc1C, E1_422, E1_399 constructs</li>
+
                         <li>Confirmed surface expression of (rsaA)-cellulase fusion proteins with Endo5A, Gluc1C, E1_422, E1_399 constructs on <i>C. crescentus</i> by SDS-PAGE and Western Blot.</li>
                         <li>Confirmed cellulase activity of Endo5A, Gluc1C, E1_422, E1_399 cellulase constructs expressed on the surface of <i>C. crescentus</i></li>
+
                         <li>Confirmed cellulase activity of Endo5A, Gluc1C, E1_422, E1_399 cellulase constructs expressed on the surface of <i>C. crescentus</i>.</li>
                        <li> Confirmed baseline intracellular cellulase activity of 4 different cellulase constructs expressed in <i>E. coli</i>: Endo5A, Gluc1C, E1, Gluc1C</li>
+
<li>Confirmed growth of <i>C. crescentus</i> strains displaying cellulases on cellulose as a sole carbon source. </li>
<li>Confirmed growth of <i>Caulobacter</i> strains displaying cellulases on celulose as a sole carbon source. </li>
+
                         <li>Codon-optimized β-1,4-endoglucanase (cenA) derived from Registry for expression in <i>C. crescentus</i>. Cloned the gene into p4A723 plasmid for the display on <i>C. crescentus</i> surface.</li></p>
                         <li>Codon-optimized β-1,4-endoglucanase (cenA) derived from Registry for expression in <i>Caulobacter</i>. Cloned the gene into p4A723 plasmid for the display on <i>Caulobacter</i> surface</li></p>
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                 </section>
 
                 </section>
  
 
                 <section id="Introduction" class="anchor">
 
                 <section id="Introduction" class="anchor">
 
                     <h2>Introduction</h2>
 
                     <h2>Introduction</h2>
                     <p>Plant biomass is the most abundant renewable resource on Earth. Usually plant biomass consists of 30-50% of cellulose, 20-40% hemicellulose carbohydrates and 20-35% lignin heteropolymer, which are integrated in each other to form complex carbohydrate  structures recalcitrant to degradation. Cellulose represents a linear polymer of glucose molecules connected through β-1,4 glycoside bonds (Fig.1). In nature the degradation of cellulose into soluble sugars often involves several different enzymes, such as exoglucanases, endoglucanases and β-glucosidases, that act in synergy to cleave the bonds.
+
                     <p>Plant biomass is the most abundant renewable resource on Earth. Usually plant biomass consists of 30-50% of cellulose, 20-40% hemicellulose carbohydrates and 20-35% lignin heteropolymer, which are integrated in each other to form complex carbohydrate  structures recalcitrant to degradation. Cellulose represents a linear polymer of glucose molecules connected through β-1,4 glycoside bonds (Fig.1). In nature the degradation of cellulose into soluble sugars often involves the synergistic action of several different enzymes, including exoglucanases, endoglucanases and β-1,4-glucosidases.
 
                     </p>
 
                     </p>
 
                     <p style="text-align:center">
 
                     <p style="text-align:center">
 
                         <img src="https://static.igem.org/mediawiki/2016/3/3e/T--British_Columbia--Cellulose.png" style="width:500px">
 
                         <img src="https://static.igem.org/mediawiki/2016/3/3e/T--British_Columbia--Cellulose.png" style="width:500px">
 
                     </p>
 
                     </p>
                     <p><b>Figure 1</b> Molecular structure of cellulose polymer.</p>
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                     <p><b>Figure 1.</b> Molecular structure of cellulose polymer.</p>
                     <p>We propose to express a mixture of cellulase-degrading enzymes on the surface of the bacteria <i>Caulobacter crescentus</i>,which will work in synergy to increase the rate of cellulose degradation and yield of monosaccharides release.</p>
+
             
                    <p>Caulobacter crescentus is a gram negative non-pathogenic bacterium found in many freshwater and
+
                     <p><i>C. crescentus</i> is a Gram negative non-pathogenic bacterium found in many freshwater and
 
                         soil environments (Poindexter,J.S. 1981). It displays, on its outer surface, a crystalline protein lattice called a surface layer (S-layer) which is made of multiple repetitions of the same RsaA protein encasing the whole surface of the cell (Fig.2)(Smit, J.1992). </p>
 
                         soil environments (Poindexter,J.S. 1981). It displays, on its outer surface, a crystalline protein lattice called a surface layer (S-layer) which is made of multiple repetitions of the same RsaA protein encasing the whole surface of the cell (Fig.2)(Smit, J.1992). </p>
 
                     <p style="text-align:center">
 
                     <p style="text-align:center">
 
                         <img src="https://static.igem.org/mediawiki/2016/d/df/T--British_Columbia--S-Layer.png" style="width:700px">
 
                         <img src="https://static.igem.org/mediawiki/2016/d/df/T--British_Columbia--S-Layer.png" style="width:700px">
 
                     </p>
 
                     </p>
                     <p><b>Figure 2</b> (left) Schematic top view on <i>Caulobacter crescentus</i> surface layer protein hexagonal arrangement
+
                     <p><b>Figure 2.</b> (left) Schematic top view on <i>C. crescentus</i> surface layer protein hexagonal arrangement
 
                         (right) Gram negative cell wall structure with the surface layer.</p>
 
                         (right) Gram negative cell wall structure with the surface layer.</p>
                     <p>For our project we have used the RsaA fusion protein secretion system designed by the Smit lab at the University of British Columbia. Using the ΔGCSS strain of <i>C.crescentus</i> which has its own wild type RsaA protein  knock-out in the host genome, we have aimed to insert genetically engineered RsaA fusion protein plasmids to express functional cellulase enzymes fused into the surface layer protein. The use of fusion proteins expressed on the surface of the cell would allow for the breakdown of cellulase without any diffusion of the substrate in and out of the cell.
+
                     <p>For our project we have used the RsaA fusion protein secretion system designed by the Smit lab at the University of British Columbia. We created a variety of cellulase-expressing <i>C. crescentus</i> strains using the S-layer as a surface expression system. We characterized expression and activity of the cellulases and tested growth of the strains on cellulose as a sole carbon source. We attempted to grow different combinations of strains to see if they could work in synergy to increase the rate of cellulose degradation and yield of monosaccharide release.</p>
                    </p>
+
  
 
                 </section>
 
                 </section>
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                 <section id="Design" class="anchor">
 
                 <section id="Design" class="anchor">
 
                     <h2>Design</h2>
 
                     <h2>Design</h2>
                     <p>WE MUST ADD THIS SECTION :)</p>
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                     <p>Engineering of the S-layer, encoded by the <i>rsaA</i> gene, has been made possible using a <i>C. crescentus</i> genomic rsaA knockout mutant, that can be complemented with a <i>rsaA</i> gene that contains an internal multiple cloning site. Using the multiple cloning site, genes encoding proteins of interest can be cloned in, in-frame, to create a construct that encodes a contiguous protein that contains the S-layer and internal protein of interest. </p>
 +
<p>
 +
In order to engineer cellulases onto the S-layer of <i>C. crescentus</i>, some key criteria had to be met for selection of candidate cellulases. This included that the cellulases had to be as small as possible, ideally less that 30 kDa, N- and C-termini within proximity of each other, and characterization in the literature for heterologous expression and activity.
 +
 
 +
Once a candidate cellulase was identified, a BLAST search against the PDB was conducted to find a structure or structural homolog. Based on the structure or PHYRE2 (Protein Homology/analogY Recognition Engine V 2.0) homology modeling, and comparison with construct design in the literature, a construct was designed (Fig 3). </p>
 +
<p style="text-align:center">
 +
                        <img src="https://static.igem.org/mediawiki/2016/4/40/British_columbia_cellulases_structures.png" style="width:1000px">
 +
                    </p>
 +
                    <p><b>Figure 3.</b> Structures of cellulases candidates.  A) Model of CenA structure generated by PHYRE2 using PDB:1UOZ, endoglucanase Cel6 from Mycobacterium tuberculosis, as a structural model. The two sequences share 42% sequence identity and the model includes amino acids 162-447 and thiocellopentose (cyan) superimposed using the substrate bound 1UOZ template structure. Colored in pale blue are amino acid residues 162-189 that were excluded from the construct design for S-layer expression. The CenA construct for S-layer expression included amino acids 190-447 and N- and C-termini are colored blue and red, respectively. B) Structure of Gluc1C bound to thiocellobiose (purple) (PDB:2O9R_A). The structure includes amino acids 4-448, which is what we chose for our construct design for S-layer expression. N- and C-termini are within close proximity (colored blue and red, respectively).  </p>
 +
 
 +
 
 +
<p>This process was done for all seven candidate cellulases. This meant that the carbohydrate domain was excluded and N-terminal regions were trimmed. Also, one cellulase, E1, constructs were designed to include (E1_422) or exclude (E1_399) the linker region between the cellulase and carbohydrate-binding domain, a P[S,T]-rich repeat region, based on activity described in the literature. Candidate cellulases were codon optimized for expression in C. crescentus and synthesized by IDT.</p>
 +
 
 +
<p>
 +
Below is a summary of candidate cellulases that were cloned into the S-layer (Table 1). We searched for beta-1,4-glucosidase, endo-glucanase, and exoglucanases in hopes that we could grow a consortia capable of completely degrading cellulose to primary glucose units. </p>
 +
 
 +
                    <div class="scrollable"><table>
 +
                        <tr>
 +
                            <th>Candidate cellulase </hd>
 +
                            <th>Organism of origin</th>
 +
<th>Enzyme type</th>
 +
<th>PDB structure (or homolog*)</th>
 +
<th>Amino acid residues included in construct</th>
 +
<th>Literature reference</th>
 +
                        </tr>
 +
                        <tr>
 +
                            <td>CEX</td>
 +
                            <td><i>Cellulomonas fimi</i></td>
 +
                            <td>exo-β-1,4-glucanase</td>
 +
                            <td>3CUF_A</td>
 +
                            <td>43-354</td>
 +
                            <td>Bingle, et al, 2000 </td>
 +
                        </tr>
 +
                        <tr>
 +
                            <td>Gluc1C</td>
 +
                            <td><i>Paenibacillus sp.</i> MTCC 5639</td>
 +
                            <td>1,4- β -glucosidase</td>
 +
                            <td>2O9R_A</td>
 +
                            <td>4-448</td>
 +
                            <td>Gupta, et al, 2013 </td>
 +
                        </tr>
 +
                        <tr>
 +
                            <td>Endo5a</td>
 +
                            <td><i>Paenibacillus sp.</i> ICGEB2008</td>
 +
                            <td>endo- β -1,4-glucanase</td>
 +
                            <td>1ECE_A*, 1VRX_A*</td>
 +
                            <td>34-385</td>
 +
                            <td>Gupta, et al, 2013 </td>
 +
                        </tr>
 +
                        <tr>
 +
                            <td>E1_399</td>
 +
                            <td><i>Acidothermus cellulolyticus</i></td>
 +
                            <td>endo- β -1,4-glucanase</td>
 +
                            <td>1ECE</td>
 +
                            <td>42-399</td>
 +
                            <td>Linger, et al, 2010 </td>
 +
                        </tr>
 +
                        <tr>
 +
                            <td>E1_422</td>
 +
                            <td><i>Acidothermus cellulolyticus</i></td>
 +
                            <td>endo- β -1,4-glucanase</td>
 +
                            <td>1ECE</td>
 +
                            <td>42-422</td>
 +
                            <td>Linger, et al, 2010 </td>
 +
                        </tr>
 +
                        <tr>
 +
                            <td>G12</td>
 +
                            <td><i>Acidothermus cellulolyticus</i></td>
 +
                            <td>endo- β -1,4-glucanase</td>
 +
                            <td>1H0B_A*</td>
 +
                            <td>35-274</td>
 +
                            <td>Linger, et al, 2010 </td>
 +
                        </tr>
 +
                        <tr>
 +
                            <td>CenA</td>
 +
                            <td><i>Cellulomonas fimi</i></td>
 +
                            <td>endo- β -1,4-glucanase</td>
 +
                            <td>1UOZ*</td>
 +
                            <td>190-447</td>
 +
                            <td>iGEM repository
 +
BBa_K118023
 +
</td>
 +
                        </tr>
 +
 
 +
                    </table></div>
 +
<p> <b>Table 1.</b> Summary of candidate cellulases that were codon optimized for C. crescentus and synthesized by IDT for cloning into the S-layer for surface expression.</p>
 
                 </section>
 
                 </section>
  
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                     <h2>Methods</h2>
 
                     <h2>Methods</h2>
 
                     <p>
 
                     <p>
                         All <i>Caulobacter</i> cultures were grown in PYE media supplemented with 2 μg/ml chloramphenicol unless
+
                         All <i>C. crescentus</i> cultures were grown in PYE media supplemented with 2 μg/ml chloramphenicol unless stated otherwise. All plasmid DNA extractions were performed with QIAprep Spin Miniprep Kit
                        stated otherwise. All plasmid DNA extractions were performed with QIAprep Spin Miniprep Kit
+
 
                         (Qiagen). DNA purification from gels or PCR mixtures were done with NucleoSpin® Gel and PCR
 
                         (Qiagen). DNA purification from gels or PCR mixtures were done with NucleoSpin® Gel and PCR
 
                         Clean-up kit(Macherey-Nagel).</p>
 
                         Clean-up kit(Macherey-Nagel).</p>
                     <h3>Cloning of cellulase enzymes into rsaA plasmid in <i>C. crescentus</i></h3>
+
                     <h3>Cloning of cellulases enzyme into rsaA plasmid in <i>C. crescentus</i></h3>
                     <p>The synthesized cellulase DNA was amplified using PCR with HF Phusion polymerase in 100 μl reactions.The following primers were used to amplify the selected region of cellulases genes and to add the BglII and PstI or NheI cut sites for cloning in p4A_723 plasmid:</p>
+
                     <p>The synthesized cellulase DNA was amplified using PCR with HF Phusion polymerase in 100 μl reactions. The following primers were used to amplify the selected region of cellulases genes and to add the BglII and PstI or NheI cut sites for cloning in p4A723 plasmid:</p>
 
                     <div class="scrollable"><table>
 
                     <div class="scrollable"><table>
 
                         <tr>
 
                         <tr>
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                     </table></div>
 
                     </table></div>
                     <p><b>Table 1</b> Primers for cloning cellulase gene inserts into rsaA P4A723 plasmid.</p>
+
                     <p><b>Table 2</b> Primers for cloning cellulase gene inserts into <i>rsaA</i> P4A723 plasmid.</p>
  
                     <p>For the cloning of Endo5A, GlucIC, G12, E1_422 and E1_399 in P4A723 rsaA plasmid, the amplified
+
                     <p>For the cloning of Endo5A, GlucIC, G12, E1_422 and E1_399 in P4A723 <i>rsaA</i> plasmid, the amplified
 
                         products and the isolated p4A723 plasmid DNA were digested with BglII and PstI restriction enzymes (NEB). For the cloning of CEX construct, the plasmid and amplified product were digested with BglII and NheI restriction enzymes. The plasmid digests were purified by agarose gel purification, while PCR product digests were purified by PCR purification. Purified digests were ligated using T4 ligase(NEB)
 
                         products and the isolated p4A723 plasmid DNA were digested with BglII and PstI restriction enzymes (NEB). For the cloning of CEX construct, the plasmid and amplified product were digested with BglII and NheI restriction enzymes. The plasmid digests were purified by agarose gel purification, while PCR product digests were purified by PCR purification. Purified digests were ligated using T4 ligase(NEB)
 
                         and the ligation mixes were then transformed in chemically competent DH5α <i>E. coli</i>.
 
                         and the ligation mixes were then transformed in chemically competent DH5α <i>E. coli</i>.
Line 228: Line 287:
 
                     <p>
 
                     <p>
 
                         After sequence confirmation, the isolated construct DNA were electroporated into electrocompetent
 
                         After sequence confirmation, the isolated construct DNA were electroporated into electrocompetent
                         <i>C.crescentus</i> and colonies were grown on a PYE-CM plate. One colony was selected and streaked
+
                         <i>C. crescentus</i> with an RsaA Amber mutation and colonies were grown on a PYE-CM plate. After 3 days of growth one colony from each plate was selected and streaked
 
                         onto a fresh plate, which would be used for all future assays.</p>
 
                         onto a fresh plate, which would be used for all future assays.</p>
  
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                     </table></div>
 
                     </table></div>
                         <p><b>Table 2</b> Primers for cloning cellulase gene inserts in pSB1C3 plasmid.</p>
+
                         <p><b>Table 3</b> Primers for cloning cellulase gene inserts in pSB1C3 plasmid.</p>
  
 
                     <p>
 
                     <p>
                         Purified pSB1C3 plasmid and the amplified Endo5A, Gluc1C, E1, G12 constructs were digested with
+
                         Purified pSB1C3 plasmid and the amplified Endo5A, Gluc1C, E1, G12 constructs were digested with
 
                         EcoRI  and SpeI restriction enzymes. The plasmid digests were purified by agarose gel purification,
 
                         EcoRI  and SpeI restriction enzymes. The plasmid digests were purified by agarose gel purification,
 
                         while PCR product digests were purified by PCR purification. Purified digests were ligated using T4
 
                         while PCR product digests were purified by PCR purification. Purified digests were ligated using T4
Line 305: Line 364:
 
                         10 mL tubes of PYE-CM were inoculated with <i>C. crescentus</i> strains carrying the different
 
                         10 mL tubes of PYE-CM were inoculated with <i>C. crescentus</i> strains carrying the different
 
                         cellulase-rsaA fusions in p4A723 plasmid as well as a positive control carrying a plasmid with wild
 
                         cellulase-rsaA fusions in p4A723 plasmid as well as a positive control carrying a plasmid with wild
                        type rsaA (P4A723) and a negative control of rsaA knock-out (ΔGCSS). Cultures were grown overnight at
+
                      type <i>rsaA</i> (P4A723) and a negative control of <i>rsaA</i> knock-out (ΔGCSS). Cultures were grown overnight at
 
                         30°C shaker. Cultures were taken out of incubator and optical density at 600 nm was measured. All
 
                         30°C shaker. Cultures were taken out of incubator and optical density at 600 nm was measured. All
 
                         cultures were then normalized to the lowest OD by diluting the remaining culture with PYE.
 
                         cultures were then normalized to the lowest OD by diluting the remaining culture with PYE.
 
                         <br>Low pH extraction (Walker et al. 1992) was performed using different pHs of HEPES buffer to
 
                         <br>Low pH extraction (Walker et al. 1992) was performed using different pHs of HEPES buffer to
                         extract only crystalline s-layer without lysing the cells. The procedure is illustrated on Fig.3. Proteins were then stored in -20°C freezer.
+
                         extract only crystalline s-layer without lysing the cells. The procedure is illustrated on Fig 4. Proteins were then stored in -20°C freezer.
 
                         Remaining cell pellet was used to prepared a cell lysate by resuspending the pellet in 150 μl of
 
                         Remaining cell pellet was used to prepared a cell lysate by resuspending the pellet in 150 μl of
 
                         lysis buffer and boiling for 5 minutes.
 
                         lysis buffer and boiling for 5 minutes.
Line 317: Line 376:
 
                             <img src="https://static.igem.org/mediawiki/2016/c/cf/T--British_Columbia--lowPH.png" style="width:700px">
 
                             <img src="https://static.igem.org/mediawiki/2016/c/cf/T--British_Columbia--lowPH.png" style="width:700px">
 
                         </p>
 
                         </p>
                         <p><b>Figure 3</b> Schematic presentation of low pH extraction procedure.</p>
+
                         <p><b>Figure 4.</b> Schematic presentation of low pH extraction procedure for removal of S-layer from <i>C. crescentus</i>.</p>
  
 
                     <h3> Surface layer fusion protein expression confirmation </h3>
 
                     <h3> Surface layer fusion protein expression confirmation </h3>
                     <p>To confirm expression of surface layer fusion protein SDS-PAGE and western blot analysis were
+
                     <p>To confirm expression of surface layer fusion protein, <i>C. crescentus</i> cultures were grown and SDS-PAGE and western blot analysis were
                         performed on extracted proteins from low pH extraction and on the cell lysates from those samples.
+
                         performed on surface proteins from low pH extraction and on the cell lysates.
 
                         SDS-PAGE was done using 8% separating gels. Coomassie Brilliant Blue staining and western
 
                         SDS-PAGE was done using 8% separating gels. Coomassie Brilliant Blue staining and western
 
                         immunoblotting was performed using protocols outlined in protocol section. Western blots were
 
                         immunoblotting was performed using protocols outlined in protocol section. Western blots were
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                         Fluorophore was detected by Odyssey Infrared Imaging System.</p>
 
                         Fluorophore was detected by Odyssey Infrared Imaging System.</p>
  
                     <h3>Caulobacter Cellulase Activity Analysis</h3>
+
                     <h3><i>C. crescentus</i> Surface Cellulase Activity Analysis</h3>
 
                     <p>
 
                     <p>
                         For cellulase enzyme activity measurement, triplicate 5mL PYE-CM starter cultures of p4A723, E1_399, E1_422, Gluc1C and Endo5A were grown
+
                         For cellulase enzyme activity measurement, triplicate 5mL PYE-CM starter cultures of p4A723 (Δ<i>rsaA C. crescentus </i> complemented with wildtype Δ<i>rsaA</i> in p4A723), E1_399, E1_422, Gluc1C and Endo5A <i>C. crescentus</i> strains were grown
 
                         in 10 mL tubes on a rotary shaker at 30°C for 2 days. Cultures were taken out of incubator and
 
                         in 10 mL tubes on a rotary shaker at 30°C for 2 days. Cultures were taken out of incubator and
 
                         OD600 was measured. All cultures were then normalized to the lowest OD 600 nm by diluting the
 
                         OD600 was measured. All cultures were then normalized to the lowest OD 600 nm by diluting the
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                     <h3>
 
                     <h3>
                         Growth of Caulobacter displaying cellulases on cellulose as a sole carbon source</h3>
+
                         Growth of <i>C. crescentus</i> displaying cellulases on cellulose as a sole carbon source</h3>
 
                         <p>
 
                         <p>
                             Triplicate 5mL PYE-CM starter cultures of p4A723, E1_399, E1_422, Gluc1C and Endo5A were grown
+
                             Triplicate 5mL PYE-CM starter cultures of P4A723 (Δ<i>rsaA C. crescentus </i> complemented with wildtype Δ<i>rsaA</i> in p4A723), E1_399, E1_422, Gluc1C and Endo5A were grown
 
                             in 10 mL tubes on a rotary shaker at 30°C for 2 days. Cultures were taken out of incubator and
 
                             in 10 mL tubes on a rotary shaker at 30°C for 2 days. Cultures were taken out of incubator and
 
                             OD600 was measured. All cultures were then normalized to the lowest OD600 by diluting the remaining
 
                             OD600 was measured. All cultures were then normalized to the lowest OD600 by diluting the remaining
 
                             cultures with PYE. 100-fold dilution was used to inoculate the cultures in 200 μL well clear plate
 
                             cultures with PYE. 100-fold dilution was used to inoculate the cultures in 200 μL well clear plate
                             containing M2 with 0.2% w/v carboxymethylcellulose(CMC). For the co-cultures, equal-volume inoculations
+
                             containing M2 with 0.2% w/v carboxymethylcellulose (CMC). For the co-cultures, equal-volume inoculations
 
                             were done to make final dilution of 100. The plate was incubate at 30C in a Tecan platereader
 
                             were done to make final dilution of 100. The plate was incubate at 30C in a Tecan platereader
 
                             and OD600 was read every 30 minutes.
 
                             and OD600 was read every 30 minutes.
Line 360: Line 419:
 
                     <h3>Cloning of cellulase enzymes into rsaA plasmid in <i>C.crescentus</i></h3>
 
                     <h3>Cloning of cellulase enzymes into rsaA plasmid in <i>C.crescentus</i></h3>
 
                     <p>
 
                     <p>
                         Endo5A, Gluc1C, E1_422, and E1_399 were all successfully cloned into the p4A723 plasmid vector and
+
                         Endo5A, Gluc1C, E1_422, and E1_399 were all successfully cloned into the p4A723 plasmid vector (Fig. 5) and
 
                         sequence confirmed by Sanger sequencing. Cloning was attempted on G12 several times, each time the
 
                         sequence confirmed by Sanger sequencing. Cloning was attempted on G12 several times, each time the
                         transformation step into DH5α E.coli failed, leading to the assumption that the gene product might
+
                         transformation step into DH5α <i>E.coli</i> failed, leading to the assumption that the gene product might
 
                         be toxic to the cell. As Endo5A, E1_422, and E1_399 are all endoglucanases, we decided to not proceed
 
                         be toxic to the cell. As Endo5A, E1_422, and E1_399 are all endoglucanases, we decided to not proceed
                         with any troubleshooting on G12. CEX gene was successfully cloned into DH5α E.coli and sequence confirmed.
+
                         with any troubleshooting on G12. CEX gene was successfully cloned into DH5α <i>E. coli</i> and sequence confirmed.
                         However after transforming the plasmid into <i>Caulobacter</i>, extreme cell clumping was observed, possibly a
+
                         However after transforming the plasmid into <i>C. crescentus</i>, extreme cell clumping was observed, possibly a
 
                         result of the CEX fusion into the S-Layer. Due to the phenotype, we did not perform functional characterization of the CEX-rsaA fusion.  
 
                         result of the CEX fusion into the S-Layer. Due to the phenotype, we did not perform functional characterization of the CEX-rsaA fusion.  
 
                     </p>
 
                     </p>
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                         <img src="https://static.igem.org/mediawiki/2016/6/6a/T--British_Columbia--plasmid_map.jpg" style="width:400px">
 
                         <img src="https://static.igem.org/mediawiki/2016/6/6a/T--British_Columbia--plasmid_map.jpg" style="width:400px">
 
                     </p>
 
                     </p>
                     <p><b>Figure 4</b> Map of rsaA p4A723 plasmid.</p>
+
                     <p><b>Figure 5.</b> Map of <i>rsaA</i> p4A723 plasmid.</p>
  
 
                     <h3>Surface layer fusion protein extraction expression confirmation</h3>
 
                     <h3>Surface layer fusion protein extraction expression confirmation</h3>
 
                     <p>Low pH extraction was done on Endo5A, Gluc1C, E1_399, and E1_422 RsaA fusion proteins as well as
 
                     <p>Low pH extraction was done on Endo5A, Gluc1C, E1_399, and E1_422 RsaA fusion proteins as well as
                         P4A732 RsaA positive control and ΔGCSS negative control. We also prepared a cell lysate from the
+
                         P4A732 RsaA positive control and ΔGCSS (Δ<i>rsaA</i>) negative control. We also prepared a cell lysate from the
 
                         remaining cell pellet to compare the amount of protein within the cell as opposed to on the surface
 
                         remaining cell pellet to compare the amount of protein within the cell as opposed to on the surface
 
                         of the cell. We ran two gels simultaneously staining one with Coomassie Brilliant blue and performing
 
                         of the cell. We ran two gels simultaneously staining one with Coomassie Brilliant blue and performing
Line 409: Line 468:
  
 
                     </table></div>
 
                     </table></div>
                     <p>On the coomassie stained gel faint bands corresponding to cellulases-rsaA fusions were observed  in
+
                     <p>On the coomassie stained gel faint bands corresponding to cellulases-RsaA fusions were observed  in
                         low PH extracted proteins. The cell lysate showed large amounts of intracellular proteins with poor
+
                         low pH extracted proteins (Figure 6B). The cell lysate showed large amounts of intracellular proteins with poor
 
                         differentiation so no conclusions could be made from these. <br>
 
                         differentiation so no conclusions could be made from these. <br>
 
                         On the western blot analysis, RsaA fusion proteins were clearly observed at higher molecular
 
                         On the western blot analysis, RsaA fusion proteins were clearly observed at higher molecular
                         weight than wild type RsaA confirming successful expression of cellulases fused to the rsaA on the surface. Nothing was displayed on the negative control showing that no wild
+
                         weight than wild type RsaA confirming successful expression of cellulases fused to the RsaA on the surface Figure 6A). No RsaA was detected in the negative control ΔGCSS (Δ<i>rsaA</i>). The cell lysate results were comparable to the low pH extracted proteins,
                        type RsaA was produced. The cell lysate results were comparable to the low pH extracted proteins,
+
 
                         with visibly more RsaA fusion proteins detectable in the cell lysate as opposed to the low pH
 
                         with visibly more RsaA fusion proteins detectable in the cell lysate as opposed to the low pH
 
                         extracted proteins. This may be a sign that not all of the fusion protein can get out of the
 
                         extracted proteins. This may be a sign that not all of the fusion protein can get out of the
Line 421: Line 479:
 
                         indicative of higher expression and secretion outside of the cell. The smaller constructs such
 
                         indicative of higher expression and secretion outside of the cell. The smaller constructs such
 
                         as E1_399 and Endo5A were seen more clearly on both the coomassie stained gel and the western blot,
 
                         as E1_399 and Endo5A were seen more clearly on both the coomassie stained gel and the western blot,
                         indicating that the smaller constructs might be easier to transport on the surface.
+
                         indicating that the smaller constructs might be easier to transport on the surface. RsaA and the fusion proteins ran about 30 kDa above expected their expected molecular weight. This could be due to incomplete denaturation of the proteins or an issue with original estimate of the size of RsaA expressed on the plasmid p4A732.  
  
                         Some fragments of lower molecular weight RsaA protein in the western, this is probably due to cleavage of the RsaA protein during the extraction.
+
                         Some fragments of lower molecular weight RsaA protein in the western, this is probably due to cleavage of the RsaA protein during the extraction. The blot was also performed with anti-serum, not purified monoclonal antibody.
 
                     </p>
 
                     </p>
 
                     <p style="text-align:center">
 
                     <p style="text-align:center">
                         <img src="https://static.igem.org/mediawiki/2016/8/89/T--British_Columbia--Gels.png" style="width:400px; height:700px">
+
                         <img src="https://static.igem.org/mediawiki/2016/b/b0/T--British_Columbia--gels.png" style="width:500px; height:700px">
 
                         </p>
 
                         </p>
 
                         <p>
 
                         <p>
                         <b>Figure 5</b> (top) Western Blot. Left to right: Thermofisher ladder, Gluc1C cell lysate, Endo5A cell lysate,
+
                         <b>Figure 6.</b> (top) Western Blot of <i>C. crescentus</I> cellulase expressing strains, ran on SDS-PAGE and blotted with anti-RsaA. Left to right: (1) Thermofisher ladder, (2) Gluc1C cell lysate, (3) Endo5A cell lysate,
                         E1_399 cell lysate, E1_422 cell lysate, ΔGCSS cell lysate, P4A723 cell lysate, Thermofisher ladder,
+
                         (4) E1_399 cell lysate, (5) E1_422 cell lysate, (6) negative control  ΔGCSS (Δ<i>rsaA</i>) cell lysate, (7) P4A723 (wildtype) cell lysate, (8) Thermofisher ladder, (9)
                         Gluc1C low pH extracted proteins, Endo5A low pH extracted proteins, E1_399 low pH extracted proteins,
+
                         Gluc1C low pH extracted proteins, (10) Endo5A low pH extracted proteins, (11) E1_399 low pH extracted proteins, (12)
                         E1_422 low pH extracted proteins, ΔGCSS low pH extracted proteins, P4A723 low pH extracted proteins
+
                         E1_422 low pH extracted proteins, (13) ΔGCSS <i>rsaA</i>) low pH extracted proteins, (14) P4A723 (wildtype) low pH extracted proteins. (bottom) SDS-PAGE stained with coomassie brilliant blue, samples loaded in the same order as above for the Western Blot.
                        <br>
+
                        <b>Figure 6</b> (bottom) Coomassie Brilliant Blue Stain. Left to right: Thermofisher ladder, Gluc1C cell lysate,
+
                        Endo5A cell lysate, E1_399 cell lysate, E1_422 cell lysate, ΔGCSS cell lysate, P4A723 cell lysate,
+
                        Thermofisher ladder, Gluc1C low pH extracted proteins, Endo5A low pH extracted proteins,
+
                        E1_399 low pH extracted proteins, E1_422 low pH extracted proteins, ΔGCSS low pH extracted proteins,
+
                        P4A723 low pH extracted proteins
+
  
 
                     </p>
 
                     </p>
                     <h3><i>Caulobacter</i> cellulase activity analysis</h3>
+
                     <h3><i>C. crescentus</i> cellulase activity analysis</h3>
 
                     <p>
 
                     <p>
                         An initial cellulase activity analysis was run using the protocols described in Method section for a period of 150 minutes. The assay mechanism is explained in Fig. 7. 2,4dinitrophenylcellobiose (DNPC) consists of cellobiose bound to a 2,4dinitrophenol chromophore. If cellulase activity is present, the release of chromophore can be observed and measured at OD400</p>
+
                         Cellulase activity analysis was run using the protocols described in Method section for a period of 150 minutes. The assay mechanism is explained in Fig. 7. 2,4-dinitrophenylcellobiose (DNPC) consists of cellobiose bound to a 2,4dinitrophenol chromophore. If cellulase activity is present, the release of chromophore can be observed and measured at OD400</p>
 
                     </p>
 
                     </p>
 
                     <p style="text-align:center">
 
                     <p style="text-align:center">
 
                         <img src="https://static.igem.org/mediawiki/2016/4/45/British_Columbia_assay.png" style="width:700px">
 
                         <img src="https://static.igem.org/mediawiki/2016/4/45/British_Columbia_assay.png" style="width:700px">
 
                     </p>
 
                     </p>
                     <p><b>Figure 7</b> Assay for cellulase activity with DNPC substrate</p>
+
                     <p><b>Figure 7.</b> Assay for cellulase activity with DNPC substrate.</p>
  
 
                     <p>
 
                     <p>
                         This assay was run on the <i>Caulobacter</i> cells expressing Endo5A, Gluc1C, E1_399, and E1_422 RsaA fusions using the wild type RsaA P4A723 protein as a control. From the first time point, cellulase-expressing <i>Caulobacters</i> demonstrated high cellulase activities, with E1_422 and E1_399 showing the highest activity level followed by Endo5A (Figure 8). Gluc1C had lower activity levels closer to that of the control, what can be explained by the glucosidase activity of Gluc1C. The enzyme cleaves a glucose molecule from DNPC first, not cellobiose what does not lead to the release of chromophore. Observation at later time points need to be taken to confirm the Gluc1C activity.
+
                         This assay was run on the <i>C. crescentus</i> cells expressing Endo5A, Gluc1C, E1_399, and E1_422 RsaA fusions using the wild type RsaA P4A723 protein as a control. From the first time point, cellulase-expressing <i>C. crescentus</i> demonstrated high cellulase activities, with E1_422 and E1_399 showing the highest activity level followed by Endo5A (Figure 8). Gluc1C had lower activity levels closer to that of the control, what can be explained by the glucosidase activity of Gluc1C. The enzyme cleaves a glucose molecule from DNPC first, not cellobiose what does not lead to the release of chromophore. Observation at later time points need to be taken to confirm the Gluc1C activity.
 
                     </p>
 
                     </p>
 
                     <p style="text-align:center">
 
                     <p style="text-align:center">
 
                         <img src="https://static.igem.org/mediawiki/2016/6/6a/British_Columbia_cellulasese_assay.png" style="width:600px">
 
                         <img src="https://static.igem.org/mediawiki/2016/6/6a/British_Columbia_cellulasese_assay.png" style="width:600px">
 
                     </p>
 
                     </p>
                     <p><b>Figure 8</b> Cellulase activity assay results for <i>Caulobacter</i> displaying cellulases</p>
+
                     <p><b>Figure 8.</b> Cellulase activity assay results for <i>C. crescentus</i> displaying cellulases compared to <i>C. crescentus</i> expressing wildtype RsaA (P4A723).</p>
<h3><i>Caulobacter</i> growth on cellulose</h3>
+
<h3><i>C. crescentus</i> growth on cellulose</h3>
 
<p>
 
<p>
<i>Caulobacter</i> cells expressing wild type RsaA, Endo5A, Gluc1C, E1_399, and E1_422 were grown on carboxymethylcellulose(CMC) as a sole carbon source to confirm the cellulase activities of the displayed enzymes. Endo5A and its co-cultures showed the best growth results on CMC (Fig.9) As <i>Caulobacter</i> has intrinsic β-1,4-glucosidase, the displayed endoglucanase cleaves the cellulose in small disaccharide, which are further transformed by periplasmic glucosidase into glucose to sustain <i>Caulobacter</i> growth. E1_399 also demonstrates growth on cellulose and interestingly when co-cultured with Gluc1C it shows synergistic growth. The results suggest <i>Caulobacter</i> can be successfully used to transform cellulose in simple sugars and confirm that the first part of our consortia works!  
+
<i>C. crescentus</i> cells expressing wild type RsaA, Endo5A, Gluc1C, E1_399, and E1_422 were grown on carboxymethylcellulose (CMC) as a sole carbon source to confirm the cellulase activities of the displayed enzymes. Endo5A and its co-cultures showed the best growth results on CMC (Fig. 9) As <i>C. crescentus</i> has intrinsic β-1,4-glucosidase, the displayed endoglucanase cleaves the cellulose in small disaccharide, which are further transformed by periplasmic glucosidase into glucose to sustain <i>C. crescentus</i> growth. E1_399 also demonstrates growth on cellulose and interestingly when co-cultured with Gluc1C it shows synergistic growth. The results suggest <i>C. crescentus</i> can be successfully used to transform cellulose in simple sugars and confirm that the first part of our consortia works!  
 
</p>
 
</p>
 
<p style="text-align:center">
 
<p style="text-align:center">
 
  <img src="https://static.igem.org/mediawiki/2016/e/e3/British_Columbia-Cellulose_growth.png" style="width:700px">
 
  <img src="https://static.igem.org/mediawiki/2016/e/e3/British_Columbia-Cellulose_growth.png" style="width:700px">
 
</p>
 
</p>
<p><b>Figure 9</b> <i>Caulobacter</i> growth on CMC over 100 hours</p>
+
<p><b>Figure 9.</b> Growth of <i>C. crescentus</i> expressing wildtype RsaA (P4A723) compared to growth of <i>C. crescentus</i> strains expressing recombinant cellulases on carboxymethylcellulose (CMC) as a sole carbon source over 100 hours.</p>
 
                 </section>
 
                 </section>
  
 
                 <section id="Conclusion" class="anchor">
 
                 <section id="Conclusion" class="anchor">
 
                     <h2>Conclusion</h2>
 
                     <h2>Conclusion</h2>
                     <p>For our project we employed robust expression and display system of <i>Caulobacter</i> S-layer for the generation of whole-cell biocatalysts. Previously, the S-layer was used to display small peptides and proteins. Our results are a first demonstration that <i>Caulobacter</i> can be successfully used for enzymes display. The cellulases enzymes used in the study were pre-selected based on their crystal structures and truncated to reduce the size without changing the catalytic activities. The expression of cellulases on cell surface was confirmed by Western blot and the cellulolytic activity was demonstrated in DNPC assay. Also we confirmed that the engineered <i>Caulobacter</i> strains can breakdown and grow on cellulose substrate.</p>
+
                     <p>For our project we employed robust expression and display system of <i>C. crescentus</i> S-layer for the generation of whole-cell biocatalysts. Previously, the S-layer was used to display small peptides and proteins. Our results are a first demonstration that <i>C. crescentus</i> can be successfully used for enzymes display. The cellulases enzymes used in the study were pre-selected based on their crystal structures and truncated to reduce the size without changing the catalytic activities. The expression of cellulases on cell surface was confirmed by Western blot and the cellulolytic activity was demonstrated in DNPC assay. Also we confirmed that the engineered <i>C. crescentus</i> strains can breakdown and grow on cellulose substrate.</p>
 
                 </section>
 
                 </section>
  
 
                 <section id="References" class="anchor">
 
                 <section id="References" class="anchor">
 
                     <h2>References</h2>
 
                     <h2>References</h2>
                     <p>Amat, F., L. R. Comolli, J. F. Nomellini, F. Moussavi, K. H. Downing, J. Smit, and M. Horowitz.
+
                     <ol>Amat, Fernando et al. “Analysis of the Intact Surface Layer of Caulobacter Crescentus by Cryo-Electron Tomography.Journal of bacteriology 192.22 (2010): 5855–65.</ol>
                    <br>    "Analysis of the Intact Surface Layer of Caulobacter Crescentus by Cryo-Electron  
+
<ol>Bingle, Wade H., John F. Nomellini, and John Smit. “Secretion of the Caulobacter Crescentus S-Layer Protein: Further Localization of the C-Terminal Secretion Signal and Its Use for Secretion of Recombinant Proteins.” Journal of Bacteriology 182.11 (2000): 3298–3301. </ol>
                    <br>    Tomography." Journal of Bacteriology 192.22 (2010): 5855-865.
+
<ol>Farr, Christina et al. “Development of an HIV-1 Microbicide Based on Caulobacter Crescentus: Blocking Infection by High-Density Display of Virus Entry Inhibitors.” PLoS ONE 8.6 (2013): </ol>
                    </p>  
+
<ol>Gandham, Lyngrace, John F Nomellini, and John Smit. “Evaluating Secretion and Surface Attachment of SapA, an S-Layer-Associated Metalloprotease of Caulobacter Crescentus.Archives of microbiology 194.10 (2012): 865–77. </ol>
                   
+
<ol>Gupta, Shefali, Nidhi Adlakha, and Syed Shams Yazdani. “2013-Efficient Extracellular Secretion of an Endoglucanase and a B-Glucosidase in E. Coli.pdf.” 88 (2013): 20–25. </ol>
                    <p>Gandham, Lyngrace, John F. Nomellini, and John Smit. "Evaluating Secretion and Surface  
+
<ol>Linger, Jeffrey G., William S. Adney, and Al Darzins. “Heterologous Expression and Extracellular Secretion of Cellulolytic Enzymes by Zymomonas Mobilis.” Applied and Environmental Microbiology 76.19 (2010): 6360–6369. </ol>
                    <br>    Attachment of SapA, an S-layer-associated Metalloprotease of Caulobacter Crescentus."
+
<ol>Mewis, Keith, Marcus Taupp, and Steven J Hallam. “A High Throughput Screen for Biomining Cellulase Activity from Metagenomic Libraries.Journal of visualized experiments : JoVE 48 (2011): n. pag. </ol>
                    <br>    Archives of Microbiology 194.10 (2012): 865-77.  
+
<ol>Poindexter, J S. “The Caulobacters: Ubiquitous Unusual Bacteria.” Microbiological reviews 45.1 (1981): 123–79. </ol>
                    </p>
+
<ol>Presley, Gerald N. et al. “Extracellular Gluco-Oligosaccharide Degradation by Caulobacter Crescentus.” Microbiology (United Kingdom) 160.PART 3 (2014): 635–645.</ol>
                   
+
<ol>Ratanakhanokchai, Khanok et al. “Paenibacillus Curdlanolyticus Strain B-6 Multienzyme Complex: A Novel System for Biomass Utilization.” Biomass Now - Cultivation and Utilization. N.p., 2013. 369–394.</ol>
                    <p>Mewis, Keith, Marcus Taupp, and Steven J. Hallam. "A High Throughput Screen for Biomining  
+
<ol>Smit, J et al. “The S-Layer of Caulobacter Crescentus: Three-Dimensional Image Reconstruction and Structure Analysis by Electron Microscopy.” Journal of bacteriology 174.20 (1992): 6527–38.</ol>
                    <br>    Cellulase Activity from Metagenomic Libraries." Journal of Visualized Experiments JoVE  
+
<ol>Taylor, Publisher. “Anti-Tumor Effects of the Bacterium Caulobacter Crescentus.” (2006): 37–41. </ol>
                    <br>     48 (2011).
+
<ol>Umelo-Njaka, Elizabeth et al. “Expression and Testing of Pseudomonas Aeruginosa Vaccine Candidate Proteins Prepared with the Caulobacter Crescentus S-Layer Protein Expression System.” Vaccine 19.11-12 (2001): 1406–1415. </ol>
                    </p>
+
<ol>Walker, S G, S H Smith, and J Smit. “Isolation and Comparison of the Paracrystalline Surface Layer Proteins of Freshwater Caulobacters.” Journal of bacteriology 174.6 (1992): 1783–92. </ol>
                   
+
                    <p>Walker, S G, S H Smith, and J Smit. “Isolation and Comparison of the Paracrystalline Surface
+
                    <br>    Layer Proteins of Freshwater Caulobacters.” Journal of Bacteriology 174.6 (1992):
+
                    <br>    1783–1792.
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Latest revision as of 02:04, 20 October 2016

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Cellulase
S-Layer Engineering

Abstract

Lignocellulosic biomass, the most abundantly available renewable resource in nature, represents a promising alternative to fossil fuels for sustainable production of chemicals, materials, and biofuels. The main technological obstacle for industrial exploitation of biomass is the recalcitrant nature of lignocellulosic polymers. One approach to harness the energy contained in biomass is to convert the lignocellulosic polymers into simple sugars, which can be further transformed in valuable compounds. Some anaerobic microorganisms have developed approaches to break down recalcitrant plant biomass using elaborate extracellular enzyme complexes, called cellulosomes, containing a scaffolding protein with many attached cellulolytic enzymes. The systems were adopted by scientists to generate artificial cellulosomes for biotechnological applications. These approaches have had their limitations due to inefficiencies with product yield, which are inevitably incurred as a consequence of the metabolic strain experienced by single microbial strains that comprise most modern bioprocessing systems.

Our approach focuses on adapting the surface layer of Caulobacter crescentus for the high density display of cellulolytic enzymes. We specifically chose C. crescentus as it natively expresses a two dimensional crystal lattice protein called RsaA, which makes up the surface layer (S-Layer). The S-layer can be engineered for surface display of recombinant proteins. Our goal was to clone cellulolytic enzymes into the S-Layer protein to create a fusion protein for surface display, granting C. crescentus the ability to degrade cellulose to simple sugars. We successfully cloned, confirmed surface expression, and activity of the cellulases. Additionally, we demonstrated that surface expression of cellulases allowed C. crescentus to grow on cellulose as a sole carbon source, compared to wild-type C. crescentus, which is unable to do so. We believe that our approach will simplify the process of "cellulosome" engineering by exploiting the robust surface display system of C. crescentus. The developed platform can be easily adapted for the display of different proteins with different enzymatic activities.

Key Achievements

  • Cloned 5 different cellulases, such as β-1,4-endoglucanase (Endo5A), β-1,4-glucosidase (Gluc1C), β-1,4-exoglucanase (CEX) and 2 versions of β-1,4-endoglucanase(E1) in p4A723 plasmid containing rsaA and transformed into C. crescentus for the display on cell surface.
  • Cloned 4 different cellulases - Endo5A, Gluc1C, E1, G12 into pSB1C3 and submitted as parts BBa_K2139001, BBa_K2139002, BBa_K2139003, BBa_K2139004.
  • Confirmed surface expression of (rsaA)-cellulase fusion proteins with Endo5A, Gluc1C, E1_422, E1_399 constructs on C. crescentus by SDS-PAGE and Western Blot.
  • Confirmed cellulase activity of Endo5A, Gluc1C, E1_422, E1_399 cellulase constructs expressed on the surface of C. crescentus.
  • Confirmed growth of C. crescentus strains displaying cellulases on cellulose as a sole carbon source.
  • Codon-optimized β-1,4-endoglucanase (cenA) derived from Registry for expression in C. crescentus. Cloned the gene into p4A723 plasmid for the display on C. crescentus surface.
  • Introduction

    Plant biomass is the most abundant renewable resource on Earth. Usually plant biomass consists of 30-50% of cellulose, 20-40% hemicellulose carbohydrates and 20-35% lignin heteropolymer, which are integrated in each other to form complex carbohydrate structures recalcitrant to degradation. Cellulose represents a linear polymer of glucose molecules connected through β-1,4 glycoside bonds (Fig.1). In nature the degradation of cellulose into soluble sugars often involves the synergistic action of several different enzymes, including exoglucanases, endoglucanases and β-1,4-glucosidases.

    Figure 1. Molecular structure of cellulose polymer.

    C. crescentus is a Gram negative non-pathogenic bacterium found in many freshwater and soil environments (Poindexter,J.S. 1981). It displays, on its outer surface, a crystalline protein lattice called a surface layer (S-layer) which is made of multiple repetitions of the same RsaA protein encasing the whole surface of the cell (Fig.2)(Smit, J.1992).

    Figure 2. (left) Schematic top view on C. crescentus surface layer protein hexagonal arrangement (right) Gram negative cell wall structure with the surface layer.

    For our project we have used the RsaA fusion protein secretion system designed by the Smit lab at the University of British Columbia. We created a variety of cellulase-expressing C. crescentus strains using the S-layer as a surface expression system. We characterized expression and activity of the cellulases and tested growth of the strains on cellulose as a sole carbon source. We attempted to grow different combinations of strains to see if they could work in synergy to increase the rate of cellulose degradation and yield of monosaccharide release.

    Design

    Engineering of the S-layer, encoded by the rsaA gene, has been made possible using a C. crescentus genomic rsaA knockout mutant, that can be complemented with a rsaA gene that contains an internal multiple cloning site. Using the multiple cloning site, genes encoding proteins of interest can be cloned in, in-frame, to create a construct that encodes a contiguous protein that contains the S-layer and internal protein of interest.

    In order to engineer cellulases onto the S-layer of C. crescentus, some key criteria had to be met for selection of candidate cellulases. This included that the cellulases had to be as small as possible, ideally less that 30 kDa, N- and C-termini within proximity of each other, and characterization in the literature for heterologous expression and activity. Once a candidate cellulase was identified, a BLAST search against the PDB was conducted to find a structure or structural homolog. Based on the structure or PHYRE2 (Protein Homology/analogY Recognition Engine V 2.0) homology modeling, and comparison with construct design in the literature, a construct was designed (Fig 3).

    Figure 3. Structures of cellulases candidates. A) Model of CenA structure generated by PHYRE2 using PDB:1UOZ, endoglucanase Cel6 from Mycobacterium tuberculosis, as a structural model. The two sequences share 42% sequence identity and the model includes amino acids 162-447 and thiocellopentose (cyan) superimposed using the substrate bound 1UOZ template structure. Colored in pale blue are amino acid residues 162-189 that were excluded from the construct design for S-layer expression. The CenA construct for S-layer expression included amino acids 190-447 and N- and C-termini are colored blue and red, respectively. B) Structure of Gluc1C bound to thiocellobiose (purple) (PDB:2O9R_A). The structure includes amino acids 4-448, which is what we chose for our construct design for S-layer expression. N- and C-termini are within close proximity (colored blue and red, respectively).

    This process was done for all seven candidate cellulases. This meant that the carbohydrate domain was excluded and N-terminal regions were trimmed. Also, one cellulase, E1, constructs were designed to include (E1_422) or exclude (E1_399) the linker region between the cellulase and carbohydrate-binding domain, a P[S,T]-rich repeat region, based on activity described in the literature. Candidate cellulases were codon optimized for expression in C. crescentus and synthesized by IDT.

    Below is a summary of candidate cellulases that were cloned into the S-layer (Table 1). We searched for beta-1,4-glucosidase, endo-glucanase, and exoglucanases in hopes that we could grow a consortia capable of completely degrading cellulose to primary glucose units.

    Candidate cellulase Organism of origin Enzyme type PDB structure (or homolog*) Amino acid residues included in construct Literature reference
    CEX Cellulomonas fimi exo-β-1,4-glucanase 3CUF_A 43-354 Bingle, et al, 2000
    Gluc1C Paenibacillus sp. MTCC 5639 1,4- β -glucosidase 2O9R_A 4-448 Gupta, et al, 2013
    Endo5a Paenibacillus sp. ICGEB2008 endo- β -1,4-glucanase 1ECE_A*, 1VRX_A* 34-385 Gupta, et al, 2013
    E1_399 Acidothermus cellulolyticus endo- β -1,4-glucanase 1ECE 42-399 Linger, et al, 2010
    E1_422 Acidothermus cellulolyticus endo- β -1,4-glucanase 1ECE 42-422 Linger, et al, 2010
    G12 Acidothermus cellulolyticus endo- β -1,4-glucanase 1H0B_A* 35-274 Linger, et al, 2010
    CenA Cellulomonas fimi endo- β -1,4-glucanase 1UOZ* 190-447 iGEM repository BBa_K118023

    Table 1. Summary of candidate cellulases that were codon optimized for C. crescentus and synthesized by IDT for cloning into the S-layer for surface expression.

    Methods

    All C. crescentus cultures were grown in PYE media supplemented with 2 μg/ml chloramphenicol unless stated otherwise. All plasmid DNA extractions were performed with QIAprep Spin Miniprep Kit (Qiagen). DNA purification from gels or PCR mixtures were done with NucleoSpin® Gel and PCR Clean-up kit(Macherey-Nagel).

    Cloning of cellulases enzyme into rsaA plasmid in C. crescentus

    The synthesized cellulase DNA was amplified using PCR with HF Phusion polymerase in 100 μl reactions. The following primers were used to amplify the selected region of cellulases genes and to add the BglII and PstI or NheI cut sites for cloning in p4A723 plasmid:

    Endo5A Endo5a_fwd 5’-TCCAGATCTAGCGTCAAGGGGTATTACCAC
    Endo5A Endo5A_rev 5’-TCCATCTGCAGACACCGGCTTCATGATCCG
    Gluc1C Gluc1C_fwd 5’-TCCAGATCTAACACGTTCATCTTTCCGGC
    Gluc1C Gluc1C_rev 5’-TCCATCTGCAGAGAACCCGTTCTTGGCCAT
    E1_399 E1_399_fwd 5’-TCCAGATCTGTTGCAGGCGGGGGTTATTG
    E1_399 E1_399_rev 5’-TCCATCTGCAGAAACCGGGTCAAATATCGATGATTTTATC
    E1_422 E1_422_fwd 5’-TCCAGATCTGTTGCAGGCGGGGGTTATTG
    E1_422 E1_422_rev 5’-TCCATCTGCAGATGAGGGGGAGGGAGAC
    G12 G12_fwd 5’-TCCAGATCTGCGACGACCTCCACG
    G12 G12_rev 5’-TCCATCTGCAGATGAGGGGGTGGGAGTAG
    CEX CEX_fwd 5’-GGGAGATCTGCGACCACGCTCAAGGAGGCCGCC
    CEX CEX_Rev 5’-CTAGCTAGCCCCGGCCGGACCGGACGTCGG

    Table 2 Primers for cloning cellulase gene inserts into rsaA P4A723 plasmid.

    For the cloning of Endo5A, GlucIC, G12, E1_422 and E1_399 in P4A723 rsaA plasmid, the amplified products and the isolated p4A723 plasmid DNA were digested with BglII and PstI restriction enzymes (NEB). For the cloning of CEX construct, the plasmid and amplified product were digested with BglII and NheI restriction enzymes. The plasmid digests were purified by agarose gel purification, while PCR product digests were purified by PCR purification. Purified digests were ligated using T4 ligase(NEB) and the ligation mixes were then transformed in chemically competent DH5α E. coli. Transformed colonies were then plated on and selected from a LB-CM plate and grown overnight in LB-CM media. Recombinant plasmid was then isolated and sent for Sanger sequencing for confirmation.

    After sequence confirmation, the isolated construct DNA were electroporated into electrocompetent C. crescentus with an RsaA Amber mutation and colonies were grown on a PYE-CM plate. After 3 days of growth one colony from each plate was selected and streaked onto a fresh plate, which would be used for all future assays.

    Cloning of cellulase enzymes into pSB1C3 plasmid with Ptac promoter and rbs

    The synthesized cellulases genes were amplified using PCR with HF Phusion polymerase in 100 μl reactions. The following primers were used to amplify the selected region of cellulases genes and to add BioBrick Prefix and Suffix:

    Endo5A Endo5A_fwd 5'-TCCGAATTCGCGGCCGCTTCTAGATGAAGAAGAAGGGC
    Endo5A Endo5A_rev 5'-TCCTACTAGTAGCGGCCGCTGCAGCTACTCCGCGGAGG
    Gluc1C Gluc1C_fwd 5'-TCCGAATTCGCGGCCGCTTCTAGATGAGCGAGAACACG
    Gluc1C Gluc1C_rev 5'-TCCTACTAGTAGCGGCCGCTGCAGTTAGAACCCGTTCTTG
    E1 E1_fwd 5'-TCCGAATTCGCGGCCGCTTCTAGATGCCTCGCGCTCT
    E1 E1_rev 5'-TCCTACTAGTAGCGGCCGCTGCAGCTAGGTGGGAGTTGGG
    G12 G12_fwd 5'-TCCGAATTCGCGGCCGCTTCTAGATGTTAGTGTTAAGAGCC
    G12 G12_rev 5'-TCCTACTAGTAGCGGCCGCTGCAGCTACGACGAAGTAGG

    Table 3 Primers for cloning cellulase gene inserts in pSB1C3 plasmid.

    Purified pSB1C3 plasmid and the amplified Endo5A, Gluc1C, E1, G12 constructs were digested with EcoRI and SpeI restriction enzymes. The plasmid digests were purified by agarose gel purification, while PCR product digests were purified by PCR purification. Purified digests were ligated using T4 ligase and the ligation mixes were then transformed in chemically competent DH5α E. coli. Transformed colonies were then plated on LB-CM plate and the isolated colonies were selected and grown overnight in LB-CM media. Recombinant plasmid was isolated and sent for Sanger sequencing for confirmation.
    pTAC was amplified from Bba_K868400 part using HF phusion PCR with the following primers to add the rbs (part Bba_B0034): Ptac_rbs_F 5'-GTTTCTTCGAATTCGCGGCCGCTTCTAGAgagctgttgacaatta, Ptac_rbs_R 5'-GTTTCTTCCTGCAGCGGCCGCTACTAGTtttctcctctttaattgttatccgctca
    The amplified pTAC and rbs part was then digested with EcoRI and SpeI restriction enzymes. pSB1C3 high copy assembly plasmid with the gene insert was digested using EcoRI and XbaI restriction enzymes. The plasmid digest was then purified by agarose gel purification while the pTAC RBS digest was purified by PCR purification. Both purified digest were ligated together using standard ligation protocol and ligation mix was then transformed in chemically competent DH5α E. coli.

    Surface layer fusion protein extraction

    10 mL tubes of PYE-CM were inoculated with C. crescentus strains carrying the different cellulase-rsaA fusions in p4A723 plasmid as well as a positive control carrying a plasmid with wild type rsaA (P4A723) and a negative control of rsaA knock-out (ΔGCSS). Cultures were grown overnight at 30°C shaker. Cultures were taken out of incubator and optical density at 600 nm was measured. All cultures were then normalized to the lowest OD by diluting the remaining culture with PYE.
    Low pH extraction (Walker et al. 1992) was performed using different pHs of HEPES buffer to extract only crystalline s-layer without lysing the cells. The procedure is illustrated on Fig 4. Proteins were then stored in -20°C freezer. Remaining cell pellet was used to prepared a cell lysate by resuspending the pellet in 150 μl of lysis buffer and boiling for 5 minutes.

    Figure 4. Schematic presentation of low pH extraction procedure for removal of S-layer from C. crescentus.

    Surface layer fusion protein expression confirmation

    To confirm expression of surface layer fusion protein, C. crescentus cultures were grown and SDS-PAGE and western blot analysis were performed on surface proteins from low pH extraction and on the cell lysates. SDS-PAGE was done using 8% separating gels. Coomassie Brilliant Blue staining and western immunoblotting was performed using protocols outlined in protocol section. Western blots were probed with primary rabbit anti-RsaA polyclonal antibodies at 1/30,000 dilution. Goat anti-rabbit IgG was used as secondary antibody at 1/50,000 dilution. Fluorophore was detected by Odyssey Infrared Imaging System.

    C. crescentus Surface Cellulase Activity Analysis

    For cellulase enzyme activity measurement, triplicate 5mL PYE-CM starter cultures of p4A723 (ΔrsaA C. crescentus complemented with wildtype ΔrsaA in p4A723), E1_399, E1_422, Gluc1C and Endo5A C. crescentus strains were grown in 10 mL tubes on a rotary shaker at 30°C for 2 days. Cultures were taken out of incubator and OD600 was measured. All cultures were then normalized to the lowest OD 600 nm by diluting the remaining culture with PYE. 1 mL of each sample was taken and resuspended with fresh PYE and spin down 3 times to remove any lysed cell debris that may interfere with cellulase activity results.

    A protocol for cellulases activity assay was adopted from Mewis K.(2015) . 150 uL of each culture were aliquoted into a clear 96 well plate(Corning) and 150 μL of assay mix (0.1 mg/ml DNPC in 50 mM pH 5.5 potassium acetate buffer) was added to the each well. OD 400 nm and OD 600 nm was measured every 30 minutes for several hours by a VarioScan plate reader(Thermo). Between measurements the culture was incubating at 30°C. Data was normalized further by dividing the OD400nm measurement by the OD600nm measurement.

    Growth of C. crescentus displaying cellulases on cellulose as a sole carbon source

    Triplicate 5mL PYE-CM starter cultures of P4A723 (ΔrsaA C. crescentus complemented with wildtype ΔrsaA in p4A723), E1_399, E1_422, Gluc1C and Endo5A were grown in 10 mL tubes on a rotary shaker at 30°C for 2 days. Cultures were taken out of incubator and OD600 was measured. All cultures were then normalized to the lowest OD600 by diluting the remaining cultures with PYE. 100-fold dilution was used to inoculate the cultures in 200 μL well clear plate containing M2 with 0.2% w/v carboxymethylcellulose (CMC). For the co-cultures, equal-volume inoculations were done to make final dilution of 100. The plate was incubate at 30C in a Tecan platereader and OD600 was read every 30 minutes.

    Results

    Cloning of cellulase enzymes into rsaA plasmid in C.crescentus

    Endo5A, Gluc1C, E1_422, and E1_399 were all successfully cloned into the p4A723 plasmid vector (Fig. 5) and sequence confirmed by Sanger sequencing. Cloning was attempted on G12 several times, each time the transformation step into DH5α E.coli failed, leading to the assumption that the gene product might be toxic to the cell. As Endo5A, E1_422, and E1_399 are all endoglucanases, we decided to not proceed with any troubleshooting on G12. CEX gene was successfully cloned into DH5α E. coli and sequence confirmed. However after transforming the plasmid into C. crescentus, extreme cell clumping was observed, possibly a result of the CEX fusion into the S-Layer. Due to the phenotype, we did not perform functional characterization of the CEX-rsaA fusion.

    Figure 5. Map of rsaA p4A723 plasmid.

    Surface layer fusion protein extraction expression confirmation

    Low pH extraction was done on Endo5A, Gluc1C, E1_399, and E1_422 RsaA fusion proteins as well as P4A732 RsaA positive control and ΔGCSS (ΔrsaA) negative control. We also prepared a cell lysate from the remaining cell pellet to compare the amount of protein within the cell as opposed to on the surface of the cell. We ran two gels simultaneously staining one with Coomassie Brilliant blue and performing western blot on the other.
    Thermo fisher prestained protein ladder was used to compare relative expected sizes of fusion proteins. Expected protein sizes were as followed:

    Protein Size
    RsaA 98 kDaltons
    Endo5A-RsaA fusion 138 kDaltons
    Gluc1C-RsaA fusion 149 kDaltons
    E1_399-RsaA fusion 138 kDaltons
    E1_422-RsaA fusion 140 kDaltons

    On the coomassie stained gel faint bands corresponding to cellulases-RsaA fusions were observed in low pH extracted proteins (Figure 6B). The cell lysate showed large amounts of intracellular proteins with poor differentiation so no conclusions could be made from these.
    On the western blot analysis, RsaA fusion proteins were clearly observed at higher molecular weight than wild type RsaA confirming successful expression of cellulases fused to the RsaA on the surface Figure 6A). No RsaA was detected in the negative control ΔGCSS (ΔrsaA). The cell lysate results were comparable to the low pH extracted proteins, with visibly more RsaA fusion proteins detectable in the cell lysate as opposed to the low pH extracted proteins. This may be a sign that not all of the fusion protein can get out of the cell and crystalize, likely due to the large size of the fusion protein. The higher visibility of some constructs over others in the low pH extraction, such as with E1_399 vs. Gluc1C, is indicative of higher expression and secretion outside of the cell. The smaller constructs such as E1_399 and Endo5A were seen more clearly on both the coomassie stained gel and the western blot, indicating that the smaller constructs might be easier to transport on the surface. RsaA and the fusion proteins ran about 30 kDa above expected their expected molecular weight. This could be due to incomplete denaturation of the proteins or an issue with original estimate of the size of RsaA expressed on the plasmid p4A732. Some fragments of lower molecular weight RsaA protein in the western, this is probably due to cleavage of the RsaA protein during the extraction. The blot was also performed with anti-serum, not purified monoclonal antibody.

    Figure 6. (top) Western Blot of C. crescentus cellulase expressing strains, ran on SDS-PAGE and blotted with anti-RsaA. Left to right: (1) Thermofisher ladder, (2) Gluc1C cell lysate, (3) Endo5A cell lysate, (4) E1_399 cell lysate, (5) E1_422 cell lysate, (6) negative control ΔGCSS (ΔrsaA) cell lysate, (7) P4A723 (wildtype) cell lysate, (8) Thermofisher ladder, (9) Gluc1C low pH extracted proteins, (10) Endo5A low pH extracted proteins, (11) E1_399 low pH extracted proteins, (12) E1_422 low pH extracted proteins, (13) ΔGCSS (ΔrsaA) low pH extracted proteins, (14) P4A723 (wildtype) low pH extracted proteins. (bottom) SDS-PAGE stained with coomassie brilliant blue, samples loaded in the same order as above for the Western Blot.

    C. crescentus cellulase activity analysis

    Cellulase activity analysis was run using the protocols described in Method section for a period of 150 minutes. The assay mechanism is explained in Fig. 7. 2,4-dinitrophenylcellobiose (DNPC) consists of cellobiose bound to a 2,4dinitrophenol chromophore. If cellulase activity is present, the release of chromophore can be observed and measured at OD400

    Figure 7. Assay for cellulase activity with DNPC substrate.

    This assay was run on the C. crescentus cells expressing Endo5A, Gluc1C, E1_399, and E1_422 RsaA fusions using the wild type RsaA P4A723 protein as a control. From the first time point, cellulase-expressing C. crescentus demonstrated high cellulase activities, with E1_422 and E1_399 showing the highest activity level followed by Endo5A (Figure 8). Gluc1C had lower activity levels closer to that of the control, what can be explained by the glucosidase activity of Gluc1C. The enzyme cleaves a glucose molecule from DNPC first, not cellobiose what does not lead to the release of chromophore. Observation at later time points need to be taken to confirm the Gluc1C activity.

    Figure 8. Cellulase activity assay results for C. crescentus displaying cellulases compared to C. crescentus expressing wildtype RsaA (P4A723).

    C. crescentus growth on cellulose

    C. crescentus cells expressing wild type RsaA, Endo5A, Gluc1C, E1_399, and E1_422 were grown on carboxymethylcellulose (CMC) as a sole carbon source to confirm the cellulase activities of the displayed enzymes. Endo5A and its co-cultures showed the best growth results on CMC (Fig. 9) As C. crescentus has intrinsic β-1,4-glucosidase, the displayed endoglucanase cleaves the cellulose in small disaccharide, which are further transformed by periplasmic glucosidase into glucose to sustain C. crescentus growth. E1_399 also demonstrates growth on cellulose and interestingly when co-cultured with Gluc1C it shows synergistic growth. The results suggest C. crescentus can be successfully used to transform cellulose in simple sugars and confirm that the first part of our consortia works!

    Figure 9. Growth of C. crescentus expressing wildtype RsaA (P4A723) compared to growth of C. crescentus strains expressing recombinant cellulases on carboxymethylcellulose (CMC) as a sole carbon source over 100 hours.

    Conclusion

    For our project we employed robust expression and display system of C. crescentus S-layer for the generation of whole-cell biocatalysts. Previously, the S-layer was used to display small peptides and proteins. Our results are a first demonstration that C. crescentus can be successfully used for enzymes display. The cellulases enzymes used in the study were pre-selected based on their crystal structures and truncated to reduce the size without changing the catalytic activities. The expression of cellulases on cell surface was confirmed by Western blot and the cellulolytic activity was demonstrated in DNPC assay. Also we confirmed that the engineered C. crescentus strains can breakdown and grow on cellulose substrate.

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