Difference between revisions of "Part:BBa K118022"
 
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The cellulolytic bacterium ''Cellulomonas fimi'' uses an exoglucanase (from ''cex'', accession M15824) along with 3 endoglucanases in the degradation of cellulose into cellobiose, before use B-glucosidase to catalyse the conversion of cellobiose to D-glucose.
 
The cellulolytic bacterium ''Cellulomonas fimi'' uses an exoglucanase (from ''cex'', accession M15824) along with 3 endoglucanases in the degradation of cellulose into cellobiose, before use B-glucosidase to catalyse the conversion of cellobiose to D-glucose.
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 +
The following are Team PuiChing_Macau 2024 contributions.
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<html>
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<p>To increase the yield of our essential oils, we aim to break down plant cell walls to allow more substances to be extracted. Thus we used β-glucosidase (P1, bglA, <a href="https://parts.igem.org/Part:BBa_K2929003">BBa_K2929003</a> from Thermotoga maritima) to hydrolyse the cello-oligosaccharides and cellobiose inside the plant cell walls into glucose monomers [1]. Moreover, we have cex from Cellulomonas fimi, P6: <a href="https://parts.igem.org/Part:BBa_K118022">BBa_K118022</a> and cex with cenA from Cellulomonas fimi, P7: BBa_K118022_<a href="https://parts.igem.org/Part:BBa_K118023">BBa_K118023</a>, composite part <a href="https://parts.igem.org/Part:BBa_K5193002">BBa_K5193002</a>, a combination of thermostable exoglucanase [3] and thermostable endoglucanase [4]. By inserting the two genes into the MCS blocks (with two T7 promoters), the engineered bacteria can therefore produce two types of enzyme in huge amounts simultaneously, providing multiple catalytic domains and enhancing efficiency [5]. We used the two enzymes to break down cellulose in plant cell walls [1], therefore releasing a greater amount of essential oil.</p>
 +
  <p>To investigate the plant cell wall degradation efficiency from our engineered bacteria, we use the DNS (3,5-dinitrosalicylic acid) method. This method allows us to access the amount of reducing sugars liberated during hydrolysis [2]. </p>
 +
  <p>After adding CMC (Carboxymethyl cellulose solution) bacteria culture, we first incubated the solution for 2 hours at room temperature, 50°C, and 90°C in order to let the reaction take place. However, we found out that the most significant impact on the result is when the incubation takes place at 50°C. (See Fig 1.) Therefore we chose to incubate the solution for 2 hours in 50°C. After adding some DNS reagent to the solution, we incubated the solution again for another 10 minutes at 50°C to stop the reaction. We then added our solution into a 96-well transparent plate for OD measurement at 540 nm. The results are shown below. </p>
 +
  <p></p> 
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  <center>
 +
    <figure>
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      <img src="https://static.igem.wiki/teams/5193/wet-lab/cellulase-dns-assay-divided-by-temperature-new.png" style="width:600px;">
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      <figcaption>Figure 1. The measured optical density absorbance after incubating at different temperatures. TBS is buffer (control) and PET11a is bacteria with empty vector (control). P1 is bglA, P6 is cex, P7 is cex _cenA.</figcaption>
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    </figure>
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  </center>
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  <br>
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    <center>
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      <figure>
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        <img src="https://static.igem.wiki/teams/5193/wet-lab/cellulase-bt-with-p.png" style="width:600px;">
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        <figcaption>Figure 2. The optical density absorbance at 540 nm of solutions with bacterial culture containing cellulose after 2 hours of incubation at 50°C. TBS is a buffer (control) and PET11a is a bacteria with an empty vector (control). P1 is bglA, P6 is cex, P7 is cex_cenA.
 +
        </figcaption>
 +
      </figure>
 +
    </center>
 +
    <p>The graph shows that the OD value of P7 exhibits the most significance, which means that it has the most impact, among the three, on the hydrolysis of the plant’s cell wall. Meanwhile, P1 stood at the second place, and P6 had the lowest OD value. All plasmids had a higher absorbency in comparison to our PET11a control.</p>
 +
  <br>
 +
    <h4>DNS over time</h4>
 +
  <p>In order to investigate our enzymes’ capacity in different incubation durations, we have conducted an overtime DNS assay. We prepared nine test tubes containing the same solution for different tests. First, we tested the OD value of the solution without any incubation. We then incubated the rest of the prepared solutions for 5, 10, 15, 30, 45, 60, 90, 120, and 150 minutes respectively. The results are shown in Figure 3.</p>
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  <br>
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  <center>
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    <figure>
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      <img src="https://static.igem.wiki/teams/5193/wet-lab/dns-over-time-0830-167p.jpg" style="width:600px;">
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      <figcaption>Figure 3. The optical density absorbance of bacterial culture solutions containing cellulase after being incubated in different time durations. </figcaption>
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    </figure>
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  </center>
 +
  <p>As shown in the graph, as the incubation time increases, the absorbance of all solutions rises until 120 minutes, at which β-glucosidase (P1) met its peak. Thus we chose to incubate the solution for 120 for any further experiments. </p>   
 +
  <br>
 +
 +
  <h4>Protein Detection Using Coomassie Blue and Western Blot</h4>
 +
  <p>To validate the expression of our desired protein, we performed SDS-PAGE (coomassie blue staining) and Western blot analysis (using FLAG tag antibody to trap our proteins).</p>
 +
  <p>Prior to performing the experiments, we added 0.5M IPTG for induction over 6 hours and 16 hours for the demonstration of the result. As seen in Figure 4 and 5, the results for all three cellulases across both induction times are similar at around 51 to 52 kDa. We, therefore, chose to utilize the 6 hour induction time for further experiments.</p>
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  <center>
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    <figure>
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      <img src="https://static.igem.wiki/teams/5193/comassie-blue.png" style="width:400px;">
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      <figcaption>Figure 4. Images of SDS-PAGE, flag-tag antibody, dyed with coomassie blue, 6 hours IPTG induction of all proteins.</figcaption>
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    </figure>
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  </center>
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  <center>
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    <figure>
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      <img src="https://static.igem.wiki/teams/5193/wet-lab/coomassie-blue-1.jpg" style="width:300px;">
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      <img src="https://static.igem.wiki/teams/5193/wet-lab/coomassie-blue-6-7.jpg" style="width:300px;">
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      <figcaption>Figure 5. Western blot with flag-tag antibody of bglA (P1), cex (P6) and cex_cenA (P7) after 6 and 16 hours of incubation.</figcaption>
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    </figure>
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  </center>
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  <br>
 +
 
 +
  <h4>Yield test</h4>
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  <p>To further validate our test results, we had done a yield test. Before the distillation process, we soaked 100g of dried lavender with our enzymes for different durations and temperatures. We first soaked the plant at room temperature for 30 minutes and measured the volume of lavender oil that is being extracted. We then soaked it at 50°C for 10 minutes and extracted the oil using distillation. The results are shown in figure 6. Moreover, to test our enzymes’ ability to improve the yield, we combined our enzymes into two groups, namely β-glucosidase (P1) with cex_cenA (P7) and therm_pelA (P3) with P7. Results are shown in Figure 6.</p> 
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  <center>
 +
    <figure>
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      <img src="https://static.igem.wiki/teams/5193/wet-lab/yield-test-with-p.png" style="width:600px;">
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      <figcaption>Figure 6. Comparison of the essential oil yield of samples treated with enzyme extracts between reacting at room temperature and at 50°C. PET11a is a bacteria with an empty vector (control). P1 is bglA, P3 is therm_pelA, P5 is pelA, P6 is cex, P7 is cex _cenA.</figcaption>
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    </figure>
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  </center>
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 +
    <br>
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    <p>It is steadily evident that in room temperature, the yield of the combination of P3 and cex_cenA (P7) is presented to be the most significant, while the combination of P1 and P7 is comparatively lower. P1, P6 and P7 are seen to have a lower yield, with P7 being the highest by having slightly beyond 1.5 mL and P6 being the lowest, having slightly over 1 mL. </p>
 +
   
 +
    <p>Moreover, after the reaction had occurred in 50C, the total volume of essential oil being extracted after reacting with the combination of P3 and P7 is shown to have the highest impact with more than 1.8 mL of essential oil, followed by the combination of P1 and P7, having 1.8 mL. In short, the combination of two enzyme extracts, P3 and P7 as well as P1 and P7, demonstrates significant improvement of oil yield. On the contrary, the volume of essential oil measured after reacting with P1, P6 and P7 respectively, is seen to have a lower yield. With P6 having the lowest yield of slightly lower than 1.2 mL; and P7 with around 1.7 mL, yielding the highest among the three plasmids. </p>
 +
   
 +
    <p>In order to choose the best reacting temperature, we also compared the yield between reacting at 50°C and at room temperature. As shown, all of the data demonstrated that the yield of extraction after being reacted at 50°C is higher than that at room temperature.</p>
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    <br>
 +
   
 +
    <p>References:</p> 
 +
    <ol>
 +
      <li>Ramani G, Meera B, Vanitha C, Rajendhran J, Gunasekaran P. Molecular cloning and expression of thermostable glucose-tolerant β-glucosidase of Penicillium funiculosum NCL1 in Pichia pastoris and its characterization. J Ind Microbiol Biotechnol. 2015 Apr;42(4):553-65. doi: 10.1007/s10295-014-1549-6. Epub 2015 Jan 28. PMID: 25626525. </li>
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      <li>Miller GL: Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 1959, 31: 426-428. 10.1021/ac60147a030</li>
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      <li>Saxena H, Hsu B, de Asis M, Zierke M, Sim L, Withers SG, Wakarchuk W. Characterization of a thermostable endoglucanase from Cellulomonas fimi ATCC484. Biochem Cell Biol. 2018 Feb;96(1):68-76. doi: 10.1139/bcb-2017-0150. Epub 2017 Oct 5. PMID: 28982013. https://pubmed.ncbi.nlm.nih.gov/28982013/</li>
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      <li>Chen, Y.-P., Hwang, I.-E., Lin, C.-J., Wang, H.-J., & Tseng, C.-P. (2012). Enhancing the stability of xylanase from Cellulomonas fimi by cell-surface display on Escherichia coli. Journal of Applied Microbiology, 112(3), 455–463. doi:10.1111/j.1365-2672.2012.05232.x</li>
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      <li>Duedu KO, French CE. Characterization of a Cellulomonas fimi exoglucanase/xylanase-endoglucanase gene fusion which improves microbial degradation of cellulosic biomass. Enzyme Microb Technol. 2016 Nov;93-94:113-121. doi: 10.1016/j.enzmictec.2016.08.005. Epub 2016 Aug 8. PMID: 27702471.</li>
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    </ol>
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</div>
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</html>
  
 
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====References====
 
====References====
 
[1]Luciano Silveira MH, Rau M, Pinto da Silva Bon E & Andreaus J. 2012. A simple and fast method for the determination of endo- and exo-cellulase activity in cellulase preparations using filter paper. Enzyme and Microbial Technology, 51: 280-285.
 
[1]Luciano Silveira MH, Rau M, Pinto da Silva Bon E & Andreaus J. 2012. A simple and fast method for the determination of endo- and exo-cellulase activity in cellulase preparations using filter paper. Enzyme and Microbial Technology, 51: 280-285.
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==Contribution ==
 
==Contribution ==
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*'''Summary:''' Enzyme activity assay (CMC-Na as substrate)  
 
*'''Summary:''' Enzyme activity assay (CMC-Na as substrate)  
 
===Characterization from iGEM19_CAU_China===
 
===Characterization from iGEM19_CAU_China===
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We linked the cex gene BBa_K118022 into a pET30a(+) backbone, then transformed this plasmid into BL21. After the overnight (about 15h) induction under the condition of 16℃ 0.08 mM, we smashed the recombinant E.coli strain with the ultrasonication to get crude enzyme solution, then we measured the enzyme activity by the method of CMC-Na(sodium carboxymethyl cellulose) assey. The result is shown on Fig.1.
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We linked the cex gene BBa_K118022 into the pET30a(+) backbone, which contains lacI sequence so that the heterogeneous proteins can be induced by IPTG. The plasmids were transferred into BL21(DE3) strain and we induced the recombinant overnight under the condition of 16℃ 0.08 mM IPTG. The expression of the fusion protein was determined by SDS-PAGE (Figure 3).
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[[File:CAU China INP fusion proteins SDS-PAGE.png|500px|thumb|center|'''Fig. 3''' SDS-PAGE assay for Cex expression, lane 2]]
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====Enzyme Activity Assay ====
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The activity of Cex was detected by measuring the cellulose degradation ability using CMC-Na as the substrate. We employed the procedures used by UESTC-China yet under the condition of citric acid-sodium citrate buffer with pH 4.8 and 50 ℃ for reaction temperature. The cells were disrupted by ultrasonication and the suspension of the centrifugal cell contents was used as the crude enzyme. We measured the cellulose degradation abilities of the crude enzyme.(Figure 4)
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[[File:CAU-China Cex activity assay.png|500px|thumb|center|'''Fig. 4'''  Activity assay for Cex crude enzyme fluid]]
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===Reference===
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[1] Z., Liu, X., Yi, L., Sun, et al.(2007) Analysis of the Current Situation of Biomass Waste Utilization in China. Environmental Science and Management, 32 (2): 104 - 106.
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[2] M., Li, C., Lin, M., Li, et al. (2016)Ice-nucleation Protein and Its Application in Bacterial surface Display Technology. Amino Acids and Biological Resources, 38 (2): 7-11.
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[3] van Bloois, E., Winter, R. T., Kolmar, H., & Fraaije, M. W. (2011). Decorating microbes: surface display of proteins on Escherichia coli. Trends in Biotechnology, 29(2), 79-86.
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==Contribution ==
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*'''Group:''' [http://2019.igem.org/Team:XMU-China iGEM Team XMU-China 2019]
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*'''Author:''' Zinuo Huang, Jisheng Xie
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*'''Summary:''' Quantitative Experiment of Exoglucanase Activity
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===Characterization from iGEM19 XMU-China===
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====Molecular weight====
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The molecular weight of cex is 47 kDa<ref>L. E. Sandercock, A. Meinke, N. R. Gilkes, D. G. Kilburn, R. A. J. Warren, Degradation of cellulases in cultures of Cellulomonas fimi. FEMS Microbiology Letters 143, 7-12 (1996).</ref>. In  Cex  enzymes, the  two  functional  domains  were  joined  by  a  hinge region  consisting solely  of  prolyl  and  threonyl  residues. The  binding  domain  was excised  from  Cex  by  proteolytic  cleavage  immediately  adjacent  to  the  carboxyl terminus  of  this hinge<ref>N. Gilkes, R. Warren, R. C. Miller, Jr., D. Kilburn, Precise excision of cellulose binding domains from two Cellulomonas fimi cellulases by a homologous protease and the effect on catalysis. The Journal of biological chemistry 263, 10401-10407 (1988).</ref>.
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====SDS-PAGE====
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This part was inserted into the expression vectors with T7 and RBS (<partinfo>BBa_K525998</partinfo>). Then the ligation mixture was transformed into ''E. coli'' DH5α, and the correct recombinant one was confirmed by chloramphenicol, colony PCR and sequencing.
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<html>
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    <figure>
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        <img src="https://2019.igem.org/wiki/images/7/7a/T--XMU-China--cex.png" height="75" style="float:center">
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        <figcaption>
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        <p style="font-size:1rem">
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        </p>
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        </figcaption>
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    </figure>
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</html>
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The constructed plasmid was transformed into ''E. coli'' BL21 (DE3). Positive clones that were selected by chloramphenicol preliminarily and then by colony PCR, while finally confirmed by sequencing were cultivated and induced by IPTG to express cellulases. The supernatant of culture, namely '''''sup''''', was obtained by centrifugation. And the total protein was gained by ultrasonication. The lysate underwent centrifugation and its supernatant, namely '''broken ''sup''''', was electrophoresed on a sodium dodecyl sulfate (SDS)-12% (wt/vol) polyacrylamide gel, followed by Coomassie blue staining (Fig. 2)
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<html>
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    <figure>
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        <img src="https://2019.igem.org/wiki/images/a/af/T--XMU-China--T7-RBS-cex_SDS-PAGE.png" height="300" style="float:center">
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        <br>
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        <figcaption>
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        <p style="font-size:1rem">
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        </p>
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        </figcaption>
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    </figure>
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</html>
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:'''Fig. 1''' SDS-PAGE analysis of protein in ''E. coli'' BL21 (DE3) cells and the medium by Coomassie blue staining. ''cex'': protein of BL21 (DE3) carrying T7-RBS-cex (linked by <partinfo>BBa_K525998</partinfo> and <partinfo>BBa_K118022</partinfo>), target bands can be seen in cells at about 47 kDa; Control: protein of BL21 (DE3) carrying T7 and RBS (<partinfo>BBa_K525998</partinfo>).
 +
 
 +
====Quantitative MUC Experiment====
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 +
Methylumbelliferyl cellobioside (MUC) in the presence of Exoglucanase is broken down into methylumbelliferone and cellobiose. Methylumbelliferone fluoresces under long wave length (λ=366 nm) ultra-violet light. Add 200 μL MUC working solution (5×) into 800 μL culture supernatant / crushed cell supernatant as reaction system. Add 200 μL MUC working solution (5×) into 800 μL LB Broth / PBS Buffer as background group. Incubate under the condition of 37 °C, 200 rpm using a shaking incubator for reaction. Take out one tube of reaction system into boiling water bath for 8 minutes to stop the reaction after interval time since reaction started. Dilute reaction samples for 100 times and pipet 200 μL diluent into Black opaque 96-well plate, measure fluorescence (Excitation 364 nm, Emission 460 nm) with TECAN<sup>®</sup> infinite M200 PRO. Using fluorescence intesity to determine the activity of Exoglucanase in test samples<ref>S. S. J. U. o. E. Lakhundi, Synthetic biology approach to cellulose degradation.  (2012).</ref>. Fig. 3 shows the results from the qualitative MUC assay.
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<html>
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    <figure>
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        <img src="https://2019.igem.org/wiki/images/e/e6/T--XMU-China--MUC.png" width="75%" style="float:center">
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        <figcaption>
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        <p style="font-size:1rem">
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        </p>
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        </figcaption>
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    </figure>
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</html>
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:'''Fig. 2'''  Assay for Quantitative Determination of cex Activity using MUC. (A) Supernatant and control. (B) Broken supernatant and control.
 +
 
 +
Assay for supernatant showed that no fluorescence intensity can be detected, which meant no enzymatic activity. But broken supernatant of culture with Cex protein can be detected fluorescence intensity.
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 +
====Reference====
 +
<references/>
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 +
=='''Usage by NFLS 2020'''==
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 +
<p>Our team focus on developing a system to turn cellulose into electricity this year. So we use Exoglucanase(cex) to break cellulose into glucose. </p>
 +
<p>SDS-PAGE was performed to ensure exoglucanase was successfully expressed, and as shown in figure1,Cex protein is in Lane 5-6. Since exoglucanase is 63.3kDa, the band suggested that exoglucanase have already been successfully expressed.</p>
 +
 
 +
[[File:T--NFLS--Figure1.png|800px|thumb|center|'''Figure 1. SDS-PAGE result of exoglucanase(cex)''']]
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 +
<p>Moreover, Congo Red assay was performed to test the activity of cex. Congo red forms red complex with interacted with cellulose. Therefore, when Congo red and cellulose were both
 +
added into the plate, the plate is red. When the cellulose in the plate is degraded into
 +
oligosaccharide or monosaccharide, the red color diminished and forming orange or transparent
 +
circle around the cell colony. Shown as figure 2, the Congo test was conducted to demonstrate that our engineered strain which contains cex is capable of degrading cellulose. From the result, we can see that together with endoglucanase(cen), cex can degrade the cellulose.
 +
</p>
 +
 
 +
[[File:T--NFLS--Figure2.png|800px|thumb|center|'''Figure 2. Congo red assay indicates the engineered bacteria contains exoglucanase(cex) can degrade cellulose''']]
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<!-- Uncomment this to enable Functional Parameter display
 
===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K118022 parameters</partinfo>
 
<partinfo>BBa_K118022 parameters</partinfo>
 
<!-- -->
 
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Latest revision as of 12:59, 2 October 2024

cex coding sequence encoding Cellulomonas fimi exoglucanase

The cellulolytic bacterium Cellulomonas fimi uses an exoglucanase (from cex, accession M15824) along with 3 endoglucanases in the degradation of cellulose into cellobiose, before use B-glucosidase to catalyse the conversion of cellobiose to D-glucose.

The following are Team PuiChing_Macau 2024 contributions.

To increase the yield of our essential oils, we aim to break down plant cell walls to allow more substances to be extracted. Thus we used β-glucosidase (P1, bglA, BBa_K2929003 from Thermotoga maritima) to hydrolyse the cello-oligosaccharides and cellobiose inside the plant cell walls into glucose monomers [1]. Moreover, we have cex from Cellulomonas fimi, P6: BBa_K118022 and cex with cenA from Cellulomonas fimi, P7: BBa_K118022_BBa_K118023, composite part BBa_K5193002, a combination of thermostable exoglucanase [3] and thermostable endoglucanase [4]. By inserting the two genes into the MCS blocks (with two T7 promoters), the engineered bacteria can therefore produce two types of enzyme in huge amounts simultaneously, providing multiple catalytic domains and enhancing efficiency [5]. We used the two enzymes to break down cellulose in plant cell walls [1], therefore releasing a greater amount of essential oil.

To investigate the plant cell wall degradation efficiency from our engineered bacteria, we use the DNS (3,5-dinitrosalicylic acid) method. This method allows us to access the amount of reducing sugars liberated during hydrolysis [2].

After adding CMC (Carboxymethyl cellulose solution) bacteria culture, we first incubated the solution for 2 hours at room temperature, 50°C, and 90°C in order to let the reaction take place. However, we found out that the most significant impact on the result is when the incubation takes place at 50°C. (See Fig 1.) Therefore we chose to incubate the solution for 2 hours in 50°C. After adding some DNS reagent to the solution, we incubated the solution again for another 10 minutes at 50°C to stop the reaction. We then added our solution into a 96-well transparent plate for OD measurement at 540 nm. The results are shown below.

Figure 1. The measured optical density absorbance after incubating at different temperatures. TBS is buffer (control) and PET11a is bacteria with empty vector (control). P1 is bglA, P6 is cex, P7 is cex _cenA.

Figure 2. The optical density absorbance at 540 nm of solutions with bacterial culture containing cellulose after 2 hours of incubation at 50°C. TBS is a buffer (control) and PET11a is a bacteria with an empty vector (control). P1 is bglA, P6 is cex, P7 is cex_cenA.

The graph shows that the OD value of P7 exhibits the most significance, which means that it has the most impact, among the three, on the hydrolysis of the plant’s cell wall. Meanwhile, P1 stood at the second place, and P6 had the lowest OD value. All plasmids had a higher absorbency in comparison to our PET11a control.


DNS over time

In order to investigate our enzymes’ capacity in different incubation durations, we have conducted an overtime DNS assay. We prepared nine test tubes containing the same solution for different tests. First, we tested the OD value of the solution without any incubation. We then incubated the rest of the prepared solutions for 5, 10, 15, 30, 45, 60, 90, 120, and 150 minutes respectively. The results are shown in Figure 3.


Figure 3. The optical density absorbance of bacterial culture solutions containing cellulase after being incubated in different time durations.

As shown in the graph, as the incubation time increases, the absorbance of all solutions rises until 120 minutes, at which β-glucosidase (P1) met its peak. Thus we chose to incubate the solution for 120 for any further experiments.


Protein Detection Using Coomassie Blue and Western Blot

To validate the expression of our desired protein, we performed SDS-PAGE (coomassie blue staining) and Western blot analysis (using FLAG tag antibody to trap our proteins).

Prior to performing the experiments, we added 0.5M IPTG for induction over 6 hours and 16 hours for the demonstration of the result. As seen in Figure 4 and 5, the results for all three cellulases across both induction times are similar at around 51 to 52 kDa. We, therefore, chose to utilize the 6 hour induction time for further experiments.

Figure 4. Images of SDS-PAGE, flag-tag antibody, dyed with coomassie blue, 6 hours IPTG induction of all proteins.
Figure 5. Western blot with flag-tag antibody of bglA (P1), cex (P6) and cex_cenA (P7) after 6 and 16 hours of incubation.

Yield test

To further validate our test results, we had done a yield test. Before the distillation process, we soaked 100g of dried lavender with our enzymes for different durations and temperatures. We first soaked the plant at room temperature for 30 minutes and measured the volume of lavender oil that is being extracted. We then soaked it at 50°C for 10 minutes and extracted the oil using distillation. The results are shown in figure 6. Moreover, to test our enzymes’ ability to improve the yield, we combined our enzymes into two groups, namely β-glucosidase (P1) with cex_cenA (P7) and therm_pelA (P3) with P7. Results are shown in Figure 6.

Figure 6. Comparison of the essential oil yield of samples treated with enzyme extracts between reacting at room temperature and at 50°C. PET11a is a bacteria with an empty vector (control). P1 is bglA, P3 is therm_pelA, P5 is pelA, P6 is cex, P7 is cex _cenA.

It is steadily evident that in room temperature, the yield of the combination of P3 and cex_cenA (P7) is presented to be the most significant, while the combination of P1 and P7 is comparatively lower. P1, P6 and P7 are seen to have a lower yield, with P7 being the highest by having slightly beyond 1.5 mL and P6 being the lowest, having slightly over 1 mL.

Moreover, after the reaction had occurred in 50C, the total volume of essential oil being extracted after reacting with the combination of P3 and P7 is shown to have the highest impact with more than 1.8 mL of essential oil, followed by the combination of P1 and P7, having 1.8 mL. In short, the combination of two enzyme extracts, P3 and P7 as well as P1 and P7, demonstrates significant improvement of oil yield. On the contrary, the volume of essential oil measured after reacting with P1, P6 and P7 respectively, is seen to have a lower yield. With P6 having the lowest yield of slightly lower than 1.2 mL; and P7 with around 1.7 mL, yielding the highest among the three plasmids.

In order to choose the best reacting temperature, we also compared the yield between reacting at 50°C and at room temperature. As shown, all of the data demonstrated that the yield of extraction after being reacted at 50°C is higher than that at room temperature.


References:

  1. Ramani G, Meera B, Vanitha C, Rajendhran J, Gunasekaran P. Molecular cloning and expression of thermostable glucose-tolerant β-glucosidase of Penicillium funiculosum NCL1 in Pichia pastoris and its characterization. J Ind Microbiol Biotechnol. 2015 Apr;42(4):553-65. doi: 10.1007/s10295-014-1549-6. Epub 2015 Jan 28. PMID: 25626525.
  2. Miller GL: Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 1959, 31: 426-428. 10.1021/ac60147a030
  3. Saxena H, Hsu B, de Asis M, Zierke M, Sim L, Withers SG, Wakarchuk W. Characterization of a thermostable endoglucanase from Cellulomonas fimi ATCC484. Biochem Cell Biol. 2018 Feb;96(1):68-76. doi: 10.1139/bcb-2017-0150. Epub 2017 Oct 5. PMID: 28982013. https://pubmed.ncbi.nlm.nih.gov/28982013/
  4. Chen, Y.-P., Hwang, I.-E., Lin, C.-J., Wang, H.-J., & Tseng, C.-P. (2012). Enhancing the stability of xylanase from Cellulomonas fimi by cell-surface display on Escherichia coli. Journal of Applied Microbiology, 112(3), 455–463. doi:10.1111/j.1365-2672.2012.05232.x
  5. Duedu KO, French CE. Characterization of a Cellulomonas fimi exoglucanase/xylanase-endoglucanase gene fusion which improves microbial degradation of cellulosic biomass. Enzyme Microb Technol. 2016 Nov;93-94:113-121. doi: 10.1016/j.enzmictec.2016.08.005. Epub 2016 Aug 8. PMID: 27702471.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 524
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 157
    Illegal NgoMIV site found at 530
    Illegal NgoMIV site found at 1032
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 577
    Illegal SapI.rc site found at 660

Contribution

  • Group: [http://2018.igem.org/Team:UESTC-China iGEM Team UESTC-China 2018]
  • Author: Liang Zhao, Yetao Zou
  • Summary: Enzyme digestion and enzyme activity assay

Characterization from iGEM18-UESTC-China

Molecular weight

This gene codes for a protein of 485 amino acids with a molecular mass of 51.2 kDa.

Enzyme digestion

We did a codon optimization of this part before using it. And we verified it by enzyme digestion.

Fig. 1Fig.1 Double enzyme digestion of BBa_K118022. Lane 1: BBa_K118022 digested by EcoRⅠ+PstⅠ. Lane 2: BBa_K118022 digested by Eco32Ⅰ+PstⅠ.

Filter paper assay

We constructed a plasmid containing cenA gene BBa_K118023, cex gene BBa_K118022. We transformed this plasmid into BL21(DE3). We used an intracellular fraction (crude enzyme solution) obtained by ultrasonication to carry out an experiment for measuring total enzyme activities by the method of filter paper assay.The result is shown on Fig. 2[1].
Fig. 2 Enzyme activity of the total cellulase at pH7.0, 40 ℃.

References

[1]Luciano Silveira MH, Rau M, Pinto da Silva Bon E & Andreaus J. 2012. A simple and fast method for the determination of endo- and exo-cellulase activity in cellulase preparations using filter paper. Enzyme and Microbial Technology, 51: 280-285.

Contribution

Characterization from iGEM19_CAU_China

We linked the cex gene BBa_K118022 into the pET30a(+) backbone, which contains lacI sequence so that the heterogeneous proteins can be induced by IPTG. The plasmids were transferred into BL21(DE3) strain and we induced the recombinant overnight under the condition of 16℃ 0.08 mM IPTG. The expression of the fusion protein was determined by SDS-PAGE (Figure 3).

Fig. 3 SDS-PAGE assay for Cex expression, lane 2

Enzyme Activity Assay

The activity of Cex was detected by measuring the cellulose degradation ability using CMC-Na as the substrate. We employed the procedures used by UESTC-China yet under the condition of citric acid-sodium citrate buffer with pH 4.8 and 50 ℃ for reaction temperature. The cells were disrupted by ultrasonication and the suspension of the centrifugal cell contents was used as the crude enzyme. We measured the cellulose degradation abilities of the crude enzyme.(Figure 4)

Fig. 4 Activity assay for Cex crude enzyme fluid


Reference

[1] Z., Liu, X., Yi, L., Sun, et al.(2007) Analysis of the Current Situation of Biomass Waste Utilization in China. Environmental Science and Management, 32 (2): 104 - 106.

[2] M., Li, C., Lin, M., Li, et al. (2016)Ice-nucleation Protein and Its Application in Bacterial surface Display Technology. Amino Acids and Biological Resources, 38 (2): 7-11.

[3] van Bloois, E., Winter, R. T., Kolmar, H., & Fraaije, M. W. (2011). Decorating microbes: surface display of proteins on Escherichia coli. Trends in Biotechnology, 29(2), 79-86.

Contribution

  • Group: [http://2019.igem.org/Team:XMU-China iGEM Team XMU-China 2019]
  • Author: Zinuo Huang, Jisheng Xie
  • Summary: Quantitative Experiment of Exoglucanase Activity

Characterization from iGEM19 XMU-China

Molecular weight

The molecular weight of cex is 47 kDa[1]. In Cex enzymes, the two functional domains were joined by a hinge region consisting solely of prolyl and threonyl residues. The binding domain was excised from Cex by proteolytic cleavage immediately adjacent to the carboxyl terminus of this hinge[2].


SDS-PAGE

This part was inserted into the expression vectors with T7 and RBS (BBa_K525998). Then the ligation mixture was transformed into E. coli DH5α, and the correct recombinant one was confirmed by chloramphenicol, colony PCR and sequencing.

The constructed plasmid was transformed into E. coli BL21 (DE3). Positive clones that were selected by chloramphenicol preliminarily and then by colony PCR, while finally confirmed by sequencing were cultivated and induced by IPTG to express cellulases. The supernatant of culture, namely sup, was obtained by centrifugation. And the total protein was gained by ultrasonication. The lysate underwent centrifugation and its supernatant, namely broken sup, was electrophoresed on a sodium dodecyl sulfate (SDS)-12% (wt/vol) polyacrylamide gel, followed by Coomassie blue staining (Fig. 2)


Fig. 1 SDS-PAGE analysis of protein in E. coli BL21 (DE3) cells and the medium by Coomassie blue staining. cex: protein of BL21 (DE3) carrying T7-RBS-cex (linked by BBa_K525998 and BBa_K118022), target bands can be seen in cells at about 47 kDa; Control: protein of BL21 (DE3) carrying T7 and RBS (BBa_K525998).

Quantitative MUC Experiment

Methylumbelliferyl cellobioside (MUC) in the presence of Exoglucanase is broken down into methylumbelliferone and cellobiose. Methylumbelliferone fluoresces under long wave length (λ=366 nm) ultra-violet light. Add 200 μL MUC working solution (5×) into 800 μL culture supernatant / crushed cell supernatant as reaction system. Add 200 μL MUC working solution (5×) into 800 μL LB Broth / PBS Buffer as background group. Incubate under the condition of 37 °C, 200 rpm using a shaking incubator for reaction. Take out one tube of reaction system into boiling water bath for 8 minutes to stop the reaction after interval time since reaction started. Dilute reaction samples for 100 times and pipet 200 μL diluent into Black opaque 96-well plate, measure fluorescence (Excitation 364 nm, Emission 460 nm) with TECAN® infinite M200 PRO. Using fluorescence intesity to determine the activity of Exoglucanase in test samples[3]. Fig. 3 shows the results from the qualitative MUC assay.

Fig. 2 Assay for Quantitative Determination of cex Activity using MUC. (A) Supernatant and control. (B) Broken supernatant and control.

Assay for supernatant showed that no fluorescence intensity can be detected, which meant no enzymatic activity. But broken supernatant of culture with Cex protein can be detected fluorescence intensity.

Reference

  1. ↑ L. E. Sandercock, A. Meinke, N. R. Gilkes, D. G. Kilburn, R. A. J. Warren, Degradation of cellulases in cultures of Cellulomonas fimi. FEMS Microbiology Letters 143, 7-12 (1996).
  2. ↑ N. Gilkes, R. Warren, R. C. Miller, Jr., D. Kilburn, Precise excision of cellulose binding domains from two Cellulomonas fimi cellulases by a homologous protease and the effect on catalysis. The Journal of biological chemistry 263, 10401-10407 (1988).
  3. ↑ S. S. J. U. o. E. Lakhundi, Synthetic biology approach to cellulose degradation. (2012).

Usage by NFLS 2020

Our team focus on developing a system to turn cellulose into electricity this year. So we use Exoglucanase(cex) to break cellulose into glucose.

SDS-PAGE was performed to ensure exoglucanase was successfully expressed, and as shown in figure1,Cex protein is in Lane 5-6. Since exoglucanase is 63.3kDa, the band suggested that exoglucanase have already been successfully expressed.

Figure 1. SDS-PAGE result of exoglucanase(cex)

Moreover, Congo Red assay was performed to test the activity of cex. Congo red forms red complex with interacted with cellulose. Therefore, when Congo red and cellulose were both added into the plate, the plate is red. When the cellulose in the plate is degraded into oligosaccharide or monosaccharide, the red color diminished and forming orange or transparent circle around the cell colony. Shown as figure 2, the Congo test was conducted to demonstrate that our engineered strain which contains cex is capable of degrading cellulose. From the result, we can see that together with endoglucanase(cen), cex can degrade the cellulose.

Figure 2. Congo red assay indicates the engineered bacteria contains exoglucanase(cex) can degrade cellulose