Difference between revisions of "Part:BBa K3394004"
 
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<partinfo>BBa_K3394004 short</partinfo>
 
<partinfo>BBa_K3394004 short</partinfo>
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Since cellulose cannot cross the membrane, we engineered E. coli to express cellulolytic complex on its surface through AIDA-1 auto-display system.
 
  
 
Our cellulolytic complex includes cellulase endo5a linked to CBD (cellulose binding domain) through a GS-linker and with a 6-histidine tag (His-tag) at the N-terminus.
 
Our cellulolytic complex includes cellulase endo5a linked to CBD (cellulose binding domain) through a GS-linker and with a 6-histidine tag (His-tag) at the N-terminus.
  
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In this design the order of genes is: first the cellulase endo5a and then CBD.
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The degrading power of our cellulolytic complex, as already mentioned in BBa_K3394000, is the cellulase enzyme Endo5a which breaks 1,4 beta glucose bonds. The other important component in our system is the CBD-cellulose binding domain. Its purpose is to bind the cellulose polymer to the displayed cellulolytic complex, thus enriching the cellulose surface with cellulose degrading bacteria, so the degradation will be more efficient.
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[[File:T--BGU-Israel--Breakwipe.jpeg|600px|thumb|center|Figure 1: decomposition of cellulose fibered wet wipes by engineered E. coli]]
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In addition, we added a 6-histidine tag (His-tag) at the N-terminus of the sequence, to label our recombinant proteins in the purification assay.
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In this design, the order of genes is: first the cellulase endo5a and then CBD.
 
We made another design in which the gene order is different: first the CBD and then the cellulase endo5a (BBa_K3394005).
 
We made another design in which the gene order is different: first the CBD and then the cellulase endo5a (BBa_K3394005).
  

Latest revision as of 12:08, 22 October 2020


Cellulolytic complex [1]: His-Endo5a-GS-CBD

Our cellulolytic complex includes cellulase endo5a linked to CBD (cellulose binding domain) through a GS-linker and with a 6-histidine tag (His-tag) at the N-terminus.

The degrading power of our cellulolytic complex, as already mentioned in BBa_K3394000, is the cellulase enzyme Endo5a which breaks 1,4 beta glucose bonds. The other important component in our system is the CBD-cellulose binding domain. Its purpose is to bind the cellulose polymer to the displayed cellulolytic complex, thus enriching the cellulose surface with cellulose degrading bacteria, so the degradation will be more efficient.

Figure 1: decomposition of cellulose fibered wet wipes by engineered E. coli

In addition, we added a 6-histidine tag (His-tag) at the N-terminus of the sequence, to label our recombinant proteins in the purification assay.

In this design, the order of genes is: first the cellulase endo5a and then CBD. We made another design in which the gene order is different: first the CBD and then the cellulase endo5a (BBa_K3394005).



Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 988
    Illegal EcoRI site found at 1314
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 988
    Illegal EcoRI site found at 1314
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 988
    Illegal EcoRI site found at 1314
    Illegal BamHI site found at 28
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 988
    Illegal EcoRI site found at 1314
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 988
    Illegal EcoRI site found at 1314
    Illegal AgeI site found at 1289
  • 1000
    COMPATIBLE WITH RFC[1000]