Difference between revisions of "Part:BBa K2324013"

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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K2324013 short</partinfo>
 
<partinfo>BBa_K2324013 short</partinfo>
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<p>
 
<p>
This part synthesises a FimH adhesin protein fused with a metallothionein at the first amino acid position. The coding sequence is under the control of a rhamnose-inducible promoter, with a B0034 RBS and a B0015 terminator. The part, when induced, produces a metal binding FimH protein that should involve itself in pilus biosynthesis when co-transformed with a plasmid containing the <i>fim operon</i> .</p>
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This part produces a FimH protein with a 6xHistidine tag inserted at the first amino acid position, that is the residue that remains at the N-terminus after the signal peptide has been cleaved during the membrane export process. This position is intended to improve the steric properties of the protein so as to ease the cell surface membrane export and to prevent interference with any native protein domains in the FimH which are involved in pilus biogenesis. The coding sequence is under the control of a rhamnose-inducible promoter, with a B0034 RBS and a B0015 terminator. The part, when induced, produces a metal binding FimH protein that should involve itself in pilus biosynthesis when co-transformed with a plasmid containing the <i>fim operon</i>  </p>
<p>
+
This part produces a FimH protein with a 6xHistidine tag inserted at the first amino acid position, that is the residue that remains at the N-terminus after the signal peptide has been cleaved during the membrane export process. This position is intended to improve the steric properties of the protein so as to ease the cell surface membrane export and to prevent interference with any native protein domains in the FimH which are involved in pilus biogenesis. </p>
+
  
<p>The part acts as a reporter. It gives clear and unambiguous evidence of protein expression. Once transformed into a number of <i>E. coli </i> strains (Top10, FimB KO, FimH KO), the fusion protein can be tested for in an induced culture (OD-0.6, [Rha]=2%) with combined use of an SDS-PAGE and a Western blot. </p>
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<p>The part acts as a reporter. It gives clear and unambiguous evidence of protein expression. Once transformed into a number of possible <i>E. coli </i> strains (Top10, FimB KO, FimH KO), the fusion protein can be expressed by  inducing the culture at 0.6 OD with 2% rhamnose for 20 hours. The production of the 6xHistidine tag can be tested by the use of an SDS-PAGE and a Western blot. </p>
  
 
<p><b>*INSERT IMAGES*</b></p>
 
<p><b>*INSERT IMAGES*</b></p>

Revision as of 16:40, 1 November 2017


pRha_FimH_1His

This part produces a FimH protein with a 6xHistidine tag inserted at the first amino acid position, that is the residue that remains at the N-terminus after the signal peptide has been cleaved during the membrane export process. This position is intended to improve the steric properties of the protein so as to ease the cell surface membrane export and to prevent interference with any native protein domains in the FimH which are involved in pilus biogenesis. The coding sequence is under the control of a rhamnose-inducible promoter, with a B0034 RBS and a B0015 terminator. The part, when induced, produces a metal binding FimH protein that should involve itself in pilus biosynthesis when co-transformed with a plasmid containing the fim operon

The part acts as a reporter. It gives clear and unambiguous evidence of protein expression. Once transformed into a number of possible E. coli strains (Top10, FimB KO, FimH KO), the fusion protein can be expressed by inducing the culture at 0.6 OD with 2% rhamnose for 20 hours. The production of the 6xHistidine tag can be tested by the use of an SDS-PAGE and a Western blot.

*INSERT IMAGES*

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 469
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 612