Difference between revisions of "Part:BBa K118022"

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*'''Group:''' [http://2019.igem.org/Team:XMU-China iGEM Team XMU-China 2019]
 
*'''Group:''' [http://2019.igem.org/Team:XMU-China iGEM Team XMU-China 2019]
 
*'''Author:''' Zinuo Huang, Jisheng Xie
 
*'''Author:''' Zinuo Huang, Jisheng Xie
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*'''Summary:''' Experiment for Quantitative Determination of Exoglucanase Activity
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*'''Summary:''' Quantitative Experiment of Exoglucanase Activity
  
 
===Characterization from iGEM19 XMU-China===
 
===Characterization from iGEM19 XMU-China===

Revision as of 11:59, 20 October 2019

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

Characterization from iGEM19_CAU_China

We linked the cex gene BBa_K118022 into the pET30a(+) backbone, which contains lacI sequence 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 IPTG. The expression of the fusion protein was determined by SDS-PAGE (Figure 3).

Fig. 3 SDS-PAGE assay for Cex expression, lane 2

Enzyme Activity Assay

The activity of Cex 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. The cells were disrupted by ultrasonication and the suspension of the centrifugal cell contents was used as the crude enzyme. We measured the cellulose degradation abilities of the crude enzyme.(Figure 4)

Fig. 4 Activity assay for Cex crude enzyme fluid

Contribution

  • Group: [http://2019.igem.org/Team:XMU-China iGEM Team XMU-China 2019]
  • Author: Zinuo Huang, Jisheng Xie
  • Summary: Quantitative Experiment of Exoglucanase Activity

Characterization from iGEM19 XMU-China

Molecular weight

The molecular weight of cex is 47 kDa[1]. In Cex enzymes, the two functional domains were joined by a hinge region consisting solely of prolyl and threonyl residues. The binding domain was excised from Cex by proteolytic cleavage immediately adjacent to the carboxyl terminus of this hinge[2].


SDS-PAGE

This part was insert into the expression vectors with T7 and RBS (BBa_K525998) by restriction sites EcoRI and PstI. Then the ligation mixture was transformed into E. coli DH5α, and the correct recombinant one was confirmed by chloramphenicol, colony PCR and sequencing.

The constructed plasmid was transformed into E. coli BL21 (DE3). Positive clones that were selected by chloramphenicol preliminarily and then by colony PCR, while finally confirmed by sequencing were cultivated and induced by IPTG to express cellulases. The supernatant of culture, namely sup, was obtained by centrifugation. And the total protein was gained by ultrasonication. The lysate underwent centrifugation and its supernatant, namely broken sup, was electrophoresed on a sodium dodecyl sulfate (SDS)-12% (wt/vol) polyacrylamide gel, followed by Coomassie blue staining (Fig. 2)


Fig. 1 SDS-PAGE analysis of protein in E. coli BL21 (DE3) cells and the medium by Coomassie blue staining. cex: protein of BL21 (DE3) carrying T7-RBS-cex (linked by BBa_K525998 and BBa_K118022), target bands can be seen in cells at about 47 kDa; Control: protein of BL21 (DE3) carrying T7 and RBS (BBa_K525998).


Quantitative MUC Experiment

Methylumbelliferyl cellobioside (MUC) in the presence of Exoglucanase is broken down into methylumbelliferone and cellobiose. Methylumbelliferone fluoresces under long wave length (λ=366 nm) ultra-violet light. Add 200 μL MUC working solution (5×) into 800 μL culture supernatant / crushed cell supernatant as reaction system. Add 200 μL MUC working solution (5×) into 800 μL LB Broth / PBS Buffer as background group. Incubate under the condition of 37 °C, 200 rpm using a shaking incubator for reaction. Take out one tube of reaction system into boiling water bath for 8 minutes to stop the reaction after interval time since reaction started. Dilute reaction samples for 100 times and pipet 200 μL diluent into Black opaque 96-well plate, measure fluorescence (Excitation 364 nm, Emission 460 nm) with TECAN® infinite M200 PRO. Using fluorescence intesity to determine the activity of Exoglucanase in test samples[3]. Fig. 3 shows the results from the qualitative MUC assay.

Fig. 2 Assay for Quantitative Determination of cex Activity using MUC. (A) Supernatant and control. (B) Broken supernatant and control.

Assay for supernatant showed that no fluorescence intensity can be detected, which meant no enzymatic activity. But broken supernatant of culture with Cex protein can be detected fluorescence intensity.

Reference

  1. ↑ L. E. Sandercock, A. Meinke, N. R. Gilkes, D. G. Kilburn, R. A. J. Warren, Degradation of cellulases in cultures of Cellulomonas fimi. FEMS Microbiology Letters 143, 7-12 (1996).
  2. ↑ N. Gilkes, R. Warren, R. C. Miller, Jr., D. Kilburn, Precise excision of cellulose binding domains from two Cellulomonas fimi cellulases by a homologous protease and the effect on catalysis. The Journal of biological chemistry 263, 10401-10407 (1988).
  3. ↑ S. S. J. U. o. E. Lakhundi, Synthetic biology approach to cellulose degradation. (2012).