Part:BBa_K4907000

sfibp-his tag

Biology

SfIBP

SfIBP is a kind of antifreeze proteins (AFPs) found from Shewanella frigidimarina (1). It is capable to prevent liquid from freezing within a certain temperature range and inhibit ice growth, which is defined as Thermal Hysteresis (TH) and ice recrystallization inhibition (IRI) respectively. Additionally, the IRI activity of SfIBP is much higher beyond other AFPs.

Usage and Design

In order to measure TH and IRI activity of SfIBP, and finally verify if it is capable to solve soil frost problem, a His-tag (6×his) was added to the C-terminal of SfIBP for purification. We constructed this part and assembled it on the expression vector pET-28a(+).

Characterization

Agarose gel electrophoresis (AGE)

The constructed plasmids were transformed into E. coli BL21(DE3), then the positive transformants were selected by kanamycin and confirmed by colony PCR and gene sequencing. Target bands (841 bp) can be observed at the position between 750 bp and 1000 bp (Fig. 1).

Fig. 1 colony PCR of BBa_K4907000_pET-28a(+) in E. coli BL21(DE3)

SDS-PAGE

The plasmid verified by sequencing was successfully transformed into E. coli BL21(DE3). After being cultivated and induced by 1 mM IPTG at 20 ℃, GE AKTA Prime Plus FPLC System was employed to get purified protein from the lysate supernatant. Purified protein was verified by sodium dodecyl sulfate (SDS)-12% (wt/vol) polyacrylamide gel electrophoresis (PAGE) and Coomassie blue staining. As shown in the gel image (Fig. 2), the target protein (25.5 kDa) can be observed at the position around 25 kDa on the purified protein lanes (FR), although displayed with many other protein bands together.

Fig. 2 SDS-PAGE analysis of SfIBP-his tag protein. Target bands (25.5 kDa) can be observed at the position around 25 kDa

Differential Scanning Calorimetry (DSC)

TH is the ability to lower freezing point (T_f) of water, which is defined as the gap between melting point (T_m) and freezing point (T_f). As Fig. 3 shows, both T_m and T_f of 50 μM SfIBP solution were measured by DSC (2), then we determined the TH activity of SfIBP.

Fig. 3 DSC curve

IRI

The least concentration of AFPs presenting IRI activity is defined as the end point of AFPs. It represented the IRI activity of AFPs, and can be detected by the capillary methods (3).

Fig. 4 IRI assay of SfIBP by the capillary method.

Characterization of soil frost resistance

Equal 50 μM SfIBP solution and Phosphate Buffered Saline (1×PBS) were added in quantitative soil to assay the ability of SfIBP to prevent soil frost.

Fig. 5 Comparison of soil added SfIBP solution and PBS

Reference

1. T. D. R. Vance, L. A. Graham, P. L. Davies, An ice-binding and tandem beta-sandwich domain-containing protein in Shewanella frigidimarina is a potential new type of ice adhesin. FEBS J. 285, 1511-1527 (2018).
2. M. Lu et al., Differential Scanning Calorimetric and Circular Dichroistic Studies on Plant Antifreeze Proteins. J. Therm. Anal. Calorim. 67, 689-698 (2002).
3. M. M. Tomczak, C. B. Marshall, J. A. Gilbert, P. L. Davies, A facile method for determining ice recrystallization inhibition by antifreeze proteins. Biochem. Biophys. Res. Commun. 311, 1041-1046 (2003).

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 AgeI site found at 634
  Illegal AgeI site found at 649
  
• 1000
  COMPATIBLE WITH RFC[1000]