Difference between revisions of "Part:BBa K118023"

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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K118023 short</partinfo>
 
<partinfo>BBa_K118023 short</partinfo>
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The cellulolytic bacterium ''Cellulomonas fimi'' uses 3 endoglucanases (including CenA, accession M15823) and an exoglucanase in the degradation of cellulose into cellobiose, before using beta-glucosidase to catalyse the conversion of cellobiose to D-glucose.
 
The cellulolytic bacterium ''Cellulomonas fimi'' uses 3 endoglucanases (including CenA, accession M15823) and an exoglucanase in the degradation of cellulose into cellobiose, before using beta-glucosidase to catalyse the conversion of cellobiose to D-glucose.
  
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This year in order to find out if cenA gene had been expressed successfully in BL21(DE3), the method of Congo Red assay was performed.  
 
This year in order to find out if cenA gene had been expressed successfully in BL21(DE3), the method of Congo Red assay was performed.  
 
Luria agar plates with 0.2% CMC were inoculated with BL21(DE3) carrying cenA gene and incubated at 37°C for 24h. After 24 h the strain was scraped off. The agar was flooded with 1 mg/ml Congo Red solution for 1h. Congo Red solution was poured off into a toxic waste bottle and 1 M NaCl was added and left for another 1 h. Then NaCl solution was poured off. Cellulases can cut CMC-Na into short chains. As Congo Red only binds to long chain polysaccharides but not short chain which therefore are washed off during staining procedure resulting in halo formation [1]. The results are shown in Fig. 1.
 
Luria agar plates with 0.2% CMC were inoculated with BL21(DE3) carrying cenA gene and incubated at 37°C for 24h. After 24 h the strain was scraped off. The agar was flooded with 1 mg/ml Congo Red solution for 1h. Congo Red solution was poured off into a toxic waste bottle and 1 M NaCl was added and left for another 1 h. Then NaCl solution was poured off. Cellulases can cut CMC-Na into short chains. As Congo Red only binds to long chain polysaccharides but not short chain which therefore are washed off during staining procedure resulting in halo formation [1]. The results are shown in Fig. 1.
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[[File:T--UESTC-China--Congo Red Assay.png|500px|thumb|center|'''Fig. 1''' Congo red assay. Line 1: BL21(DE3) carrying cenA gene. Line 2: Positive control enzyme.  Line 3: BL21(DE3) carrying empty vector control]]
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[[File:T--UESTC-China--Congo Red Assay.png|500px|thumb|center|'''Fig. 1''' Activity determination of cellulase using Congo Red assay. (a) CMC agar plate before staining with Congo Red. (b)CMC agar plate after staining with Congo Red. Module001: BL21(DE3) carrying piGEM2018-Module001; Positive Control: commercial cellulase; Negative Control: BL21 (DE3) carrying empty vector]]
 
The strains carrying cenA showed a zone of clearance created by hydrolysis of CMC. The empty vector control didn't show any zone of clearance around the colonies. The results showed that cenA gene can successfully works in BL21(DE3).
 
The strains carrying cenA showed a zone of clearance created by hydrolysis of CMC. The empty vector control didn't show any zone of clearance around the colonies. The results showed that cenA gene can successfully works in BL21(DE3).
  
 
====Endoglucanase activity assay ====
 
====Endoglucanase activity assay ====
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In addition, we measured the release of reducing sugar from CMC-Na with the 3,5-dinitrosalicylic acid (DNS) method for endoglucanase activity[2].   
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In addition, we measured the release of reducing sugar from CMC-Na with the 3,5-dinitrosalicylic acid (DNS) method for endoglucanase activity [2].   
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  [[File:T--UESTC-China-- CMCase activity assay.png|500px|thumb|center|'''Fig. 2''' endoglucanase activity assay]]
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  [[File:T--UESTC-China-- CMCase activity assay.png|500px|thumb|center|'''Fig. 2''' endoglucanase activity assay (pH7.0 40℃)]]
 
As shown in Fig. 2, BL21(DE3) carrying cenA gene could decompose CMC-Na while negative control couldn't, which proved that cenA could work successfully.
 
As shown in Fig. 2, BL21(DE3) carrying cenA gene could decompose CMC-Na while negative control couldn't, which proved that cenA could work successfully.
  
 
====References====
 
====References====
 +
[1] Lakhundi, S. S. (2012). Synthetic biology approach to cellulose degradation.
  
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[2] Wood TM & Bhat KM. 1988. Methods for measuring cellulase activities. Methods in Enzymology, 160: 87-112.
  
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==Further Improvement==
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*'''Group:''' [http://2019.igem.org/Team:CAU-China iGEM Team CAU-China 2019]
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*'''Author:''' Siwen Liang and Xueshan Yan
  
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==== Overview ====
 +
We improved this part by fusing with INP-N ( New part [https://parts.igem.org/Part:BBa_K3279008 BBa_K3279008]), the INP-N fused endoglucanase (INPN-CenA) can anchor in the cell membrane and function for surface display. The fusion protein was expressed and confirmed by SDS-PAGE, and the anchoring effect is measured through immunofluorescence staining and enzyme activity assay.For more details please see the Experience page of this part.
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  
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<partinfo>BBa_K118023 parameters</partinfo>
 
<partinfo>BBa_K118023 parameters</partinfo>
 
<!-- -->
 
<!-- -->
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==Contribution ==
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<br>Group: SYSU-CHINA 2021
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<br>Author: Zhao Bingnan
 +
<br>Summary: 3D structure predicted by swiss-model;codon-optimized cenA
 +
<br>Uploads:CenA_3D_stucture_predicted_by_swiss-model.png
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<br>[[Image:CenA 3D stucture predicted by swiss-model.png | border | center | 300px]]
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<br><font size="1"><center>Figure 1.cenA 3D structure predicted by swiss-model</center></font>
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<br>We also create a codon-optimized new part to improve the expression efficiency of the cenA, whose registry number is BBa_K3960013.For more information,please visit the main page.

Latest revision as of 02:53, 14 October 2021

cenA coding sequence encoding Cellulomonas fimi endoglucanase A The cellulolytic bacterium Cellulomonas fimi uses 3 endoglucanases (including CenA, accession M15823) and an exoglucanase in the degradation of cellulose into cellobiose, before using beta-glucosidase to catalyse the conversion of cellobiose to D-glucose.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 34
    Illegal NotI site found at 1178
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1091
    Illegal BamHI site found at 227
    Illegal XhoI site found at 589
    Illegal XhoI site found at 838
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 369
    Illegal NgoMIV site found at 1294
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 273

Contribution

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

Characterization from iGEM18-UESTC-China

Molecular weight

This gene codes for a protein of 448 amino acids with a molecular mass of 46.7 kDa.

Congo Red Assay

This year in order to find out if cenA gene had been expressed successfully in BL21(DE3), the method of Congo Red assay was performed. Luria agar plates with 0.2% CMC were inoculated with BL21(DE3) carrying cenA gene and incubated at 37°C for 24h. After 24 h the strain was scraped off. The agar was flooded with 1 mg/ml Congo Red solution for 1h. Congo Red solution was poured off into a toxic waste bottle and 1 M NaCl was added and left for another 1 h. Then NaCl solution was poured off. Cellulases can cut CMC-Na into short chains. As Congo Red only binds to long chain polysaccharides but not short chain which therefore are washed off during staining procedure resulting in halo formation [1]. The results are shown in Fig. 1.

Fig. 1 Activity determination of cellulase using Congo Red assay. (a) CMC agar plate before staining with Congo Red. (b)CMC agar plate after staining with Congo Red. Module001: BL21(DE3) carrying piGEM2018-Module001; Positive Control: commercial cellulase; Negative Control: BL21 (DE3) carrying empty vector

The strains carrying cenA showed a zone of clearance created by hydrolysis of CMC. The empty vector control didn't show any zone of clearance around the colonies. The results showed that cenA gene can successfully works in BL21(DE3).

Endoglucanase activity assay

In addition, we measured the release of reducing sugar from CMC-Na with the 3,5-dinitrosalicylic acid (DNS) method for endoglucanase activity [2].

Fig. 2 endoglucanase activity assay (pH7.0 40℃)

As shown in Fig. 2, BL21(DE3) carrying cenA gene could decompose CMC-Na while negative control couldn't, which proved that cenA could work successfully.

References

[1] Lakhundi, S. S. (2012). Synthetic biology approach to cellulose degradation.

[2] Wood TM & Bhat KM. 1988. Methods for measuring cellulase activities. Methods in Enzymology, 160: 87-112.

Further Improvement

  • Group: [http://2019.igem.org/Team:CAU-China iGEM Team CAU-China 2019]
  • Author: Siwen Liang and Xueshan Yan

Overview

We improved this part by fusing with INP-N ( New part BBa_K3279008), the INP-N fused endoglucanase (INPN-CenA) can anchor in the cell membrane and function for surface display. The fusion protein was expressed and confirmed by SDS-PAGE, and the anchoring effect is measured through immunofluorescence staining and enzyme activity assay.For more details please see the Experience page of this part.


Contribution


Group: SYSU-CHINA 2021
Author: Zhao Bingnan
Summary: 3D structure predicted by swiss-model;codon-optimized cenA
Uploads:CenA_3D_stucture_predicted_by_swiss-model.png


CenA 3D stucture predicted by swiss-model.png

Figure 1.cenA 3D structure predicted by swiss-model


We also create a codon-optimized new part to improve the expression efficiency of the cenA, whose registry number is BBa_K3960013.For more information,please visit the main page.