Difference between revisions of "Part:BBa K3279007"
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<partinfo>BBa_K3279007 short</partinfo>
 
<partinfo>BBa_K3279007 short</partinfo>
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K3279007 SequenceAndFeatures</partinfo>
  
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&#946;-Glucosidase obtained from various sources has been used in many areas, such as the enzymatic saccharification of cellulosic materials, the liberation of flavor compounds in fruit juices and wines, and so on. In cellulose hydrolysis, &#946;-glucosidase is used to degrade cello-oligosaccharides to glucose[1]. We used this part to accomplish the cellulose degradation.
 
  
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β-Glucosidase obtained from various sources has been used in many areas, such as the enzymatic saccharification of cellulosic materials, the liberation of flavor compounds in fruit juices and wines and so on. In cellulose degradation, β-glucosidase is used to break cello-oligosaccharides into glucose. We use this part to accomplish the last step of cellulose degradation. The gene encoding β-glucosidase was obtained by PCR amplification from the genomic DNA of Streptomyces coelicolor.
  
 
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===Usage and Biology===
 
===Usage and Biology===
  
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We linked the β-glucosidase coding sequence into the pET30a(+) backbone, which contains lacI sequence so that the heterogeneous proteins can be induced by IPTG, then transferred this plasmid into BL21(DE3)strain. After the overnight (about 15h) induction under the condition of 16℃ 0.08 mM IPTG, the recombinant cells are disrupted by ultrasonication to obtain the crude enzyme. We measured the enzyme activity by the method of CMC-Na(sodium carboxymethyl cellulose) assay. Considering the relatively weak cellulose degradation capacity of β-glucosidase, we mixed the crude enzyme fluid with endoglucanase([https://parts.igem.org/Part:BBa_K118023 BBa_K118023]) crude enzyme fluid. The result is shown on Fig.2.
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<span class='h3bb'>Sequence and Features</span>
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[[File:CAU-China beta-glucosidase touchdown PCR.png|500px|thumb|center|'''Fig. 1''' beta-glucosidase touchdown PCR]]
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<partinfo>BBa_K3279007 SequenceAndFeatures</partinfo>
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[[File:CAU-China INP mixed cellulases activity assays.png|500px|thumb|center|'''Fig. 2''' beta-glucosidase activity assay, the control group is 100μL buffer+ 100μL CenA(endoglucanase A from Cellulomonas fimi and the experimental group is 100μL beta-glucosidase+ 100μL CenA)]]
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===Reference===
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Hsu, C.-L., Chang, K.-S., Lai, M.-Z., Chang, T.-C., Chang, Y.-H., & Jang, H.-D. (2011). Pretreatment and hydrolysis of cellulosic agricultural wastes with a cellulase-producing Streptomyces for bioethanol production. Biomass and Bioenergy, 35, 1878-1884.
  
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We linked β-glucosidase coding sequence into a pET30a(+) backbone, then transformed this plasmid into BL21. After the overnight (about 15h) induction under the condition of 16℃ 0.08 mM（see Fig.1[1]）, 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) assay. Considering the relatively weak cellulose degradation capacity of β-glucosidase, we mixed the crude enzyme solution with endoglucanase. The result is shown on Fig.2[1].
 
  
  

Latest revision as of 03:44, 22 October 2019


β-Glucosidase from Streptomyces coelicolor Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 287
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 427
    Illegal NgoMIV site found at 1103
    Illegal AgeI site found at 184
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 687
    Illegal BsaI.rc site found at 946


β-Glucosidase obtained from various sources has been used in many areas, such as the enzymatic saccharification of cellulosic materials, the liberation of flavor compounds in fruit juices and wines and so on. In cellulose degradation, β-glucosidase is used to break cello-oligosaccharides into glucose. We use this part to accomplish the last step of cellulose degradation. The gene encoding β-glucosidase was obtained by PCR amplification from the genomic DNA of Streptomyces coelicolor.

Usage and Biology

We linked the β-glucosidase coding sequence into the pET30a(+) backbone, which contains lacI sequence so that the heterogeneous proteins can be induced by IPTG, then transferred this plasmid into BL21(DE3)strain. After the overnight (about 15h) induction under the condition of 16℃ 0.08 mM IPTG, the recombinant cells are disrupted by ultrasonication to obtain the crude enzyme. We measured the enzyme activity by the method of CMC-Na(sodium carboxymethyl cellulose) assay. Considering the relatively weak cellulose degradation capacity of β-glucosidase, we mixed the crude enzyme fluid with endoglucanase(BBa_K118023) crude enzyme fluid. The result is shown on Fig.2.

Fig. 1 beta-glucosidase touchdown PCR
Fig. 2 beta-glucosidase activity assay, the control group is 100μL buffer+ 100μL CenA(endoglucanase A from Cellulomonas fimi and the experimental group is 100μL beta-glucosidase+ 100μL CenA)

Reference

Hsu, C.-L., Chang, K.-S., Lai, M.-Z., Chang, T.-C., Chang, Y.-H., & Jang, H.-D. (2011). Pretreatment and hydrolysis of cellulosic agricultural wastes with a cellulase-producing Streptomyces for bioethanol production. Biomass and Bioenergy, 35, 1878-1884.