Coding

Part:BBa_K3279007

Designed by: Liang Siwen   Group: iGEM19_CAU_China   (2019-10-15)
Revision as of 15:43, 20 October 2019 by Milkweed (Talk | contribs)


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 hydrolysis, β-glucosidase is used to degrade cello-oligosaccharides to glucose[1]. We used this part to accomplish the cellulose degradation.


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)


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


[edit]
Categories
Parameters
None