Difference between revisions of "Part:BBa K2324008"
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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 258 of the mature FimH protein.</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 258 of the mature FimH protein.</p> | ||
<p> | <p> | ||
| − | This part produces a FimH adhesin protein fused with sfGFP at its 258th amino acid residue after signal peptide cleavage. The coding sequence is under the control of a rhamnose-inducible promoter, with a B0034 RBS and a B0015 terminator. The part, when induced, should produce a fluorescent FimH protein that should initiate pilus biosynthesis when co-transformed with a plasmid containing the <i>fim operon</i | + | This part produces a FimH adhesin protein fused with sfGFP (Pedelacq et al 2005) at its 258th amino acid residue after signal peptide cleavage. The coding sequence is under the control of a rhamnose-inducible promoter, with a B0034 RBS and a B0015 terminator. The part, when induced, should produce a fluorescent FimH protein that should initiate pilus biosynthesis when co-transformed with a plasmid containing the <i>fim operon</i>. Unfortunately despite best efforts (multiple growth temperatures and multiple rhamnose inducing concentrations) no fluorescence has been detected in any of the <i>E. coli</i> strains. </p> |
| + | <h2>References </h2> | ||
| + | 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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| + | 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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| + | Pédelacq, J.-D., Cabantous, S., Tran, T., and Terwilliger, T. C. (2005) Engineering and characterization of a superfolder green fluorescent protein. Nature Biotechnology 24, 79–88. | ||
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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. | ||
<!-- Add more about the biology of this part here | <!-- Add more about the biology of this part here | ||
===Usage and Biology=== | ===Usage and Biology=== | ||
Latest revision as of 21:50, 1 November 2017
pRha_FimH_258sfGFP
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 258 of the mature FimH protein.
This part produces a FimH adhesin protein fused with sfGFP (Pedelacq et al 2005) at its 258th amino acid residue after signal peptide cleavage. The coding sequence is under the control of a rhamnose-inducible promoter, with a B0034 RBS and a B0015 terminator. The part, when induced, should produce a fluorescent FimH protein that should initiate pilus biosynthesis when co-transformed with a plasmid containing the fim operon. Unfortunately despite best efforts (multiple growth temperatures and multiple rhamnose inducing concentrations) no fluorescence has been detected in any of the E. coli strains.
References
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.
Pédelacq, J.-D., Cabantous, S., Tran, T., and Terwilliger, T. C. (2005) Engineering and characterization of a superfolder green fluorescent protein. Nature Biotechnology 24, 79–88.
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]