Difference between revisions of "Part:BBa K2324009"
 
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<partinfo>BBa_K2324009 short</partinfo>
 
<partinfo>BBa_K2324009 short</partinfo>
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<p>The literature has shown that the terminal pili protein FimH (Le Trong et al 2010) can be modified by inserting heterologous sequences at position 225 and 258 (Pallesen et al 1995, Shembri et al 1999). However these two amino acids are in the pilin binding domain which may present difficulties when attempting to introduce large modifications. Harvard iGEM 2015 also introduced modifications at position 1 of the mature FimH protein.</p>
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<p>
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This part codes for a FimH adhesin fused with a metallothionein protein from <i>Synechococcus elongatus </i> (Blindauer et al 20011) at amino acid residue 1, after signal peptide cleavage. It is designed to be placed on the end of a type 1 pilus structure in <i> E. coli </i> with a view to binding cadmium and zinc metal ions. The coding sequence (BBa_K2324004) is under the control of a rhamnose-inducible promoter (BBa_K902065), with the BBa_ B0034 RBS and the BBa_B0015 terminator. The part, when induced, produces a FimH+metal binding protein that should initiate pilus biosynthesis when co-transformed with a plasmid containing the <i>fim operon</i>. Unfortunately despite best efforts we have been unable to characterise this part. </p>
  
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This part synthesises a FimH adhesin protein fused with a metallothionein domain at the first amino acid position. The coding sequence (BBa_K2324004) is under the control of a rhamnose-inducible promoter, with a B0034 RBS and a B0015 terminator. The part, when induced, produces a FimH+metal binding protein that should involve itself in pilus biosynthesis when co-transformed with a plasmid containing the fim operon .
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<h2>References </h2>
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Blindauer, C. A., Harrison, M. D., Parkinson, J. A., Robinson, A. K., Cavet, J. S., Robinson, N. J., and Sadler, P. J. (2001) A metallothionein containing a zinc finger within a four-metal cluster protects a bacterium from zinc toxicity. Proc. Natl. Acad. Sci. U.S.A. 98, 9593–9598.  
  
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<!-- Add more about the biology of this part here
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Le Trong, I., Aprikian, P., Kidd, B. A., Forero-Shelton, M., Tchesnokova, V., Rajagopal, P., Rodriguez, V., Interlandi, G., Klevit, R., Vogel, V., Stenkamp, R. E., Sokurenko, E. V., and Thomas, W. E. (2010) Structural Basis for Mechanical Force Regulation of the Adhesin FimH via Finger Trap-like Sheet Twisting. Cell 141, 645–655.
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===Usage and Biology===
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Pallesen, L., Poulsen, L. K., Christiansen, G., and Klemm, P. (1995) Chimeric Fimh Adhesin of Type-1 Fimbriae - a Bacterial Surface Display System for Heterologous Sequences. Microbiology 141, 2839–2848.
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Schembri, M. A., Kjaergaard, K., and KLEMM, P. (1999) Bioaccumulation of heavy metals by fimbrial designer adhesins. FEMS Microbiology Letters 170, 363–371.
  
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K2324009 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K2324009 SequenceAndFeatures</partinfo>

Latest revision as of 22:05, 1 November 2017


pRha_FimH_1SynMT

The literature has shown that the terminal pili protein FimH (Le Trong et al 2010) can be modified by inserting heterologous sequences at position 225 and 258 (Pallesen et al 1995, Shembri et al 1999). However these two amino acids are in the pilin binding domain which may present difficulties when attempting to introduce large modifications. Harvard iGEM 2015 also introduced modifications at position 1 of the mature FimH protein.

This part codes for a FimH adhesin fused with a metallothionein protein from Synechococcus elongatus (Blindauer et al 20011) at amino acid residue 1, after signal peptide cleavage. It is designed to be placed on the end of a type 1 pilus structure in E. coli with a view to binding cadmium and zinc metal ions. The coding sequence (BBa_K2324004) is under the control of a rhamnose-inducible promoter (BBa_K902065), with the BBa_ B0034 RBS and the BBa_B0015 terminator. The part, when induced, produces a FimH+metal binding protein that should initiate pilus biosynthesis when co-transformed with a plasmid containing the fim operon. Unfortunately despite best efforts we have been unable to characterise this part.

References

Blindauer, C. A., Harrison, M. D., Parkinson, J. A., Robinson, A. K., Cavet, J. S., Robinson, N. J., and Sadler, P. J. (2001) A metallothionein containing a zinc finger within a four-metal cluster protects a bacterium from zinc toxicity. Proc. Natl. Acad. Sci. U.S.A. 98, 9593–9598.

Le Trong, I., Aprikian, P., Kidd, B. A., Forero-Shelton, M., Tchesnokova, V., Rajagopal, P., Rodriguez, V., Interlandi, G., Klevit, R., Vogel, V., Stenkamp, R. E., Sokurenko, E. V., and Thomas, W. E. (2010) Structural Basis for Mechanical Force Regulation of the Adhesin FimH via Finger Trap-like Sheet Twisting. Cell 141, 645–655.

Pallesen, L., Poulsen, L. K., Christiansen, G., and Klemm, P. (1995) Chimeric Fimh Adhesin of Type-1 Fimbriae - a Bacterial Surface Display System for Heterologous Sequences. Microbiology 141, 2839–2848.


Schembri, M. A., Kjaergaard, K., and KLEMM, P. (1999) Bioaccumulation of heavy metals by fimbrial designer adhesins. FEMS Microbiology Letters 170, 363–371.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1145
    Illegal AgeI site found at 572
  • 1000
    COMPATIBLE WITH RFC[1000]