Difference between revisions of "Part:BBa K4137009"
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<partinfo>BBa_K4137009 short</partinfo>
 
<partinfo>BBa_K4137009 short</partinfo>
  
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This part consists of a T7 strong promoter, RBS (AAGGAG), a motor protein sequence yebF (BBa_K2922002), a GS Linker (BBa_J18921), a Chi19H8 coding sequence, a 6xHis tag (GTG x6), and a terminator (BBa_B1006). This part synthesizes and secretes a novel bacterial chitinase Chi18H8, which can be released extracellularly through e.coli Sec pathway (SecB-SecA-SecYEG, OmpF). This system continuously promotes the overexpression of Chi18H8 to lyse the chitin cell wall of the fungus fusarium oxysporum f.sp cubense, inhibiting fungal proliferation.
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This part synthesizes and secretes a novel bacterial chitinase Chi18H8, which can be released extracellularly through e.coli Sec pathway (SecB-SecA-SecYEG, OmpF). This system continuously promotes the overexpression of Chi18H8 to lyse the chitin cell wall of the fungus fusarium oxysporum f.sp cubense, inhibiting fungal proliferation.
  
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[[File:t7-yebf-gs-chitinase.png|800px|thumb|center|Fig.1 Chitinase (chi18h8) SEC secretion construct.]]
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===Usage and Biology===
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===Construct Designs===
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We attached a 6x His-Tag downstream of the chi18h8 sequence for purification purposes. Attached to the upstream of the chi18h8 sequence is a glycine-serine linker (GS linker) (BBa_J18921) that links chi18h8 to a yebF secretion tag (BBa_K2922002), which forms our open reading frame (ORF). We used a T7 strong promoter and flanked RBS (AAGGAG) upstream of the ORF. The terminator BBa_B1006 is attached downstream of the ORF.
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===Characterization===
  
 
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Revision as of 05:49, 9 October 2022


T7+RBS+YebF-GS Linker-chi18h8-6xHis+B1006

This part synthesizes and secretes a novel bacterial chitinase Chi18H8, which can be released extracellularly through e.coli Sec pathway (SecB-SecA-SecYEG, OmpF). This system continuously promotes the overexpression of Chi18H8 to lyse the chitin cell wall of the fungus fusarium oxysporum f.sp cubense, inhibiting fungal proliferation.

Fig.1 Chitinase (chi18h8) SEC secretion construct.

Construct Designs

We attached a 6x His-Tag downstream of the chi18h8 sequence for purification purposes. Attached to the upstream of the chi18h8 sequence is a glycine-serine linker (GS linker) (BBa_J18921) that links chi18h8 to a yebF secretion tag (BBa_K2922002), which forms our open reading frame (ORF). We used a T7 strong promoter and flanked RBS (AAGGAG) upstream of the ORF. The terminator BBa_B1006 is attached downstream of the ORF.

Characterization

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1523
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1036
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 583
  • 1000
    COMPATIBLE WITH RFC[1000]