Difference between revisions of "Part:BBa K118023"
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==Further Improvement==
 
==Further Improvement==
 
*'''Group:''' [http://2019.igem.org/Team:CAU-China iGEM Team CAU-China 2019]
 
*'''Group:''' [http://2019.igem.org/Team:CAU-China iGEM Team CAU-China 2019]
 +
*'''Author:''' Siwen Liang and Xueshan Yan
  
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We improved this part by fusing with INP-N (see [https://parts.igem.org/Part:BBa_K3279008 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. 6His tag was added to the original and fusion protein 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, 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).  
+
==== Overview ====
 +
We improved this part by fusing with INP-N (see [https://parts.igem.org/Part:BBa_K3279008 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.
 +
[[File:CAU China INP fusion proteins SDS-PAGE.png|500px|thumb|center|'''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).  
 +
[[File:CAU-China_INP_fusion_protein_20x_fluorescence_microscopy.png|750px|thumb|center|'''Fig. 4'''  E.coli cell immunofluorescence staining observation via 20x objective]]
 +
[[File:CAU-China INP fusion protein 100x confocal fluorescence microscopy.png|750px|thumb|center|'''Fig. 5'''  E.coli cell immunofluorescence staining observation via 100x objective]]
 
Under the same condition of 20X magnification and 355ms for exposure, we noted that the fluorescent signals of the original 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 some cells, while we didn`t observe this phenomenon in the field of the original 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 cell membrane or between the cell membrane and the cell wall.   
 
Under the same condition of 20X magnification and 355ms for exposure, we noted that the fluorescent signals of the original 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 some cells, while we didn`t observe this phenomenon in the field of the original 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 cell membrane or between the cell membrane and the cell wall.   
−
  [[File:CAU China INP fusion proteins SDS-PAGE.png|600px|thumb|center|'''Fig. 3''' SDS-PAGE assay for fusion protein]]
+
   
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<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  
 
===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K118023 parameters</partinfo>
 
<partinfo>BBa_K118023 parameters</partinfo>
 
<!-- -->
 
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Revision as of 17:14, 18 October 2019

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 20x objective
Fig. 5 E.coli cell immunofluorescence staining observation via 100x objective

Under the same condition of 20X magnification and 355ms for exposure, we noted that the fluorescent signals of the original 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 some cells, while we didn`t observe this phenomenon in the field of the original 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 cell membrane or between the cell membrane and the cell wall.