Part:BBa_K118022

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.

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

• Group: iGEM Team CAU-China 2019
• Author: Liang siwen, Yan xueshan
• Summary: Enzyme activity assay (CMC-Na as substrate)

Characterization from iGEM19_CAU_China

We linked the cex gene BBa_K118022 into a pET30a(+) backbone, which contains lacZ fragment 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. The expression of the fusion protein was determined by SDS-PAGE (Figure 3). and the anchoring effect is measured through immunofluorescence staining and enzyme activity assay. We fused the INP with both Cex and CenA([1]), which is the twin part of Cex, and conducted similar procedures to both parts.

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.