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 (see BBa_K3279008), the INP-N fused endoglucanase (INP-CenA) can anchor in the cell membrane and function for surface display. The fusion protein was expressed and confirmed by SDS-PAGE(Figure 3), and the anchoring effect is measured through immunofluorescence staining and enzyme activity assay. We fused the INP with both CenA and Cex and conducted similar procedures.

Fig. 3 SDS-PAGE assay for fusion protein

Microscopy Observation

6His tag was added to the original protein CenA sequence as well as the fusion protein INP-CenA as the antigen to be targeted by the primary antibody. Logically, since the CenA-6His is originally expressed in the interior of the cell, we would not detect the fluorescence in the sample of CenA and Cex, while the fluorescence is detectable for INP-CenA-6His due to the cell membrane anchoring effect. We observed the E.coli cells expressing the original proteins (CenA and Cex)and the fusion proteins (INP-CenA and INP-Cex)under the fluorescence microscopy`s 20X objective (Figure 4) and confocal fluorescence microscopy`s 100X objective (Figure 5).

Fig. 4 E.coli cell immunofluorescence staining observation via fluorescence_microscopy 20x objective

Fig. 5 E.coli cell immunofluorescence staining observation via confocal fluorescence microscopy, 100x objective (images are local zoomed)

Under the same condition of 20X magnification and 355ms for exposure, we noted that the fluorescent signals of the unfused protein field are dimmer than those of the fusion protein field on average. To examine it more clearly, we observed the slices with the confocal fluorescence microscopy. The field of fusion protein samples showed that some foci are located on the borders of the cells, while this phenomenon was not observed in the field of the unfused protein samples. But due to the minuscule size of E.coli cells, our equipment falls short when trying to determine whether the fluorescent dot on a single cell is located on the outer membrane surface or not.

Fusion enzyme activity assay

The effect of fusion on enzyme activity 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. We measured the cellulose degradation abilities of the supernatant of disrupted cell contents as well as the undisrupted cell suspensions. According to the standard curve of glucose concentration, we determined the activities of unfused enzymes CenA and Cex, and fusion enzymes INP-CenA and INP-Cex.(Figure 6)

Fig. 6 Enzyme activities assay (pH4.8 50℃)

From the data, we summarized that the cellulases` activities were not affected remarkably with the presence of INP-N. Also, the difference of enzyme activities between the ultrasonic-disrupted samples and undisrupted samples provided another evidence of the anchoring effect of INP-N. Since the fusion protein is anchored in the outer membrane surface, which would appear in the sediments after centrifugation，the samples of suspension with fused cellulases showed the relatively low level of the activity, compared with samples of unfused ones.