Part:BBa_K118023

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

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.