Difference between revisions of "Part:BBa K118022"

Revision as of 14:38, 16 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

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, then transformed this plasmid into BL21(DE3). The enzyme is induced to express under the condition of 16℃ overnight with 0.08 mM IPTG. We smashed the recombinant E.coli cells with the ultrasonication to obtain the crude enzyme solution, then we measured the enzyme activity by the method of CMC-Na(sodium carboxymethyl cellulose) assay. The expression result is shown in Fig.3.

@@ Line 33: / Line 33: @@
 *'''Summary:''' Enzyme activity assay (CMC-Na as substrate)
 ===Characterization from iGEM19_CAU_China===
-We linked the cex gene BBa_K118022 into a pET30a(+) backbone, then transformed this plasmid into BL21. After the overnight (about 15h) induction under the condition of 16℃ 0.08 mM, we smashed the recombinant E.coli strain with the ultrasonication to get crude enzyme solution, then we measured the enzyme activity by the method of CMC-Na(sodium carboxymethyl cellulose) assey. The result is shown on Fig.1.
+We linked the cex gene BBa_K118022 into a pET30a(+) backbone, then transformed this plasmid into BL21(DE3). The enzyme is induced to express under the condition of 16℃ overnight with 0.08 mM IPTG. We smashed the recombinant E.coli cells with the ultrasonication to obtain the crude enzyme solution, then we measured the enzyme activity by the method of CMC-Na(sodium carboxymethyl cellulose) assay. The expression result is shown in Fig.3.
 ===Functional Parameters===
 <partinfo>BBa_K118022 parameters</partinfo>
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