Part:BBa_K5199004

INP Silicatein

Ice Nucleation Protein(INP)- Silicatein Fusion Protein

INP is a transmembrane protein that acts as an external display marker. INP fusion proteins can be successfully expressed on the outside of E. coli. INP has been used by other teams (K409000 and K4578009), here we combined INP with a truncated silicatein alpha gene (BBa_K5199003) which polymerizes and precipitate silica. With external silicatein expression, we are able to precipitate silica on the outside of E. coli.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 321
  Illegal XhoI site found at 1246
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 63
  Illegal NgoMIV site found at 396
  Illegal AgeI site found at 498
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 765
  Illegal SapI.rc site found at 229
  

IR Spectroscopy Characterization

In order to verify the polymerization of silicon dioxide bonds by E. coli INP-sil, we performed IR spectroscopy. We also used IR spectroscopy to verify the production of an amorphous aluminosilicate in the presence of aluminum.

Figure 1: IR of E. coli INP-sil. There is an alcohol group at a broad peak around 3400cm^-1. This alcohol group is coupled with silicon, making the alcohol more diluted and not as strong of a peak. The peak is shown by a Si-O-H annotated on the spectra. There is a minor peak around 1630cm^-1, which is water within the Si-O-Si structure (Ellerbrock). Additionally, the peaks from around 1100cm^-1 to 500cm^-1 signal the presence of Si-O-Si alone. Suggests the effective polymerization of silica by E. coli INP-sil.

Figure 2: IR of E. coli INP-sil and aluminum.There is an alcohol group at a broad peak around 3400cm^-1. This alcohol group is coupled with silicon, making the alcohol more diluted and not as strong of a peak. There is a minor peak around 1630cm^-1, which is water within the Si-O-Si structure (Ellerbrock). Additionally, the peak from around 1100cm^-1 signals the presence of Si-O-Si alone. Around the 500 to 1500cm^-1 range shows the different types of peaks within the structures O−Si−O and Al−O−Si (Treto-Suarez). The most notable peak in this range is Al-O-Si around 500 to 1000cm^-1. There are two peaks around 1630cm^-1 to 1330cm^-1, which is attributed to water within the Si-O-Si structure (Ellerbrock). These peaks are indicative of an amorphous hydrated aluminum silicate, suggesting the production of an amorphous aluminosilicate by E. coli INP-sil in an aluminum rich environment.

Together, these IR spectroscopy results suggest the INP Silicatein composite part is effective at polymerizing silica in E. coli, and capable of incorporating aluminum into the polymerization process.

Sources

Ellerbrock, R., Stein, M. & Schaller, J. Comparing amorphous silica, short-range-ordered silicates and silicic acid species by FTIR. Sci Rep 12, 11708 (2022). https://doi.org/10.1038/s41598-022-15882-4.

Treto-Suarez, Manuel A. & Prieto-García, Julio & Mollineda-Trujillo, Ángel & Lamazares Arcia, Emilio & Hidalgo-Rosa, Yoan & Mena Ulecia, Karel. (2020). Kinetic study of removal heavy metal from aqueous solution using the synthetic aluminum silicate. Scientific Reports. 10. 10836. 10.1038/s41598-020-67720-0.