Difference between revisions of "Part:BBa K3110047"
 
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FimH protein is a subunit of type 1 pili present in E coli. The FimH adhesin found at the tip of type 1 pili binds naturally to mannose. Substitution of 1 amino acid at the 136th position disrupts its ability to bind to mannose. Introduction of the above mutation and adding RPMrel, a colon cancer homing peptide downstream ensures it selective binding to colon cancer cells. This part was already developed with an arabinose promoter, a SpyTag and a HisTag downstream to  RPMrel. We amplified the FimHRPMrel from this existing part, and to this, we ligated a strong constitutive promoter and strong RBS for greater expression.  
 
FimH protein is a subunit of type 1 pili present in E coli. The FimH adhesin found at the tip of type 1 pili binds naturally to mannose. Substitution of 1 amino acid at the 136th position disrupts its ability to bind to mannose. Introduction of the above mutation and adding RPMrel, a colon cancer homing peptide downstream ensures it selective binding to colon cancer cells. This part was already developed with an arabinose promoter, a SpyTag and a HisTag downstream to  RPMrel. We amplified the FimHRPMrel from this existing part, and to this, we ligated a strong constitutive promoter and strong RBS for greater expression.  
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===Usage and Biology===
 
  
 
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<partinfo>BBa_K3110047 parameters</partinfo>
 
<partinfo>BBa_K3110047 parameters</partinfo>
 
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<h1>Characterization </h1>
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After several  failed attempts, we successfully developed the above construct by SOEing. The construct was digested and ligated into pSB1C3 and transformed into DH5alpha, but no positive colonies were obtained on colony PCR. Due to time constraints, this construct was not pursued further and hence we could not characterise this construct.

Latest revision as of 12:29, 21 October 2019


Promoter RBS FimH 136KO-RPMrel

FimH protein is a subunit of type 1 pili present in E coli. The FimH adhesin found at the tip of type 1 pili binds naturally to mannose. Substitution of 1 amino acid at the 136th position disrupts its ability to bind to mannose. Introduction of the above mutation and adding RPMrel, a colon cancer homing peptide downstream ensures it selective binding to colon cancer cells. This part was already developed with an arabinose promoter, a SpyTag and a HisTag downstream to RPMrel. We amplified the FimHRPMrel from this existing part, and to this, we ligated a strong constitutive promoter and strong RBS for greater expression.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 549
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]



Characterization

After several failed attempts, we successfully developed the above construct by SOEing. The construct was digested and ligated into pSB1C3 and transformed into DH5alpha, but no positive colonies were obtained on colony PCR. Due to time constraints, this construct was not pursued further and hence we could not characterise this construct.