Difference between revisions of "Part:BBa K4907000"
 
(10 intermediate revisions by 3 users not shown)
Line 4: Line 4:
  
  
−
====Biology====
+
===Biology===
 +
====SfIBP====
 +
SfIBP is a kind of antifreeze proteins (AFPs) found from <i>Shewanella frigidimarina</i> (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 <I>E. coli</i> 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).
  
−
<center><html><img src="https://static.igem.wiki/teams/4907/wiki/parts/niao.png" width="400px"></html></center>
+
<center><html><img src="https://static.igem.wiki/teams/4907/wiki/parts/yyn/fig-1-bba-k4907000-sfibp-his-tag.png" width="400px"></html></center>
 +
<center>Fig. 1 colony PCR of BBa_K4907000_pET-28a(+) in <I>E. coli</i> BL21(DE3)</center>
  
−
====sfibp====
+
====SDS-PAGE====
−
SfIBP is a kind of antifreeze proteins (AFPs) found from <i>Shewanella frigidimarina</i> (1). It is capable to prevent liquid from freezing within a certain temperature range and inhibit ice growth, which is defined as <b>Thermal Hysteresis (TH)</b> and <b>ice recrystallization inhibition (IRI)</b> respectively. Additionally, the IRI activity of SfIBP is much higher beyond other AFPs.
+
The plasmid verified by sequencing was successfully transformed into <i>E. coli</i> 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.
  
−
===Usage and Design===
+
<center><html><img src="https://static.igem.wiki/teams/4907/wiki/parts/yyn/fig-2-bba-k4907000-sfibp-his-tag.png" width="400px"></html></center>
−
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(+).
+
<center>Fig. 2 SDS-PAGE analysis of SfIBP-his tag protein. Target bands (25.5 kDa) can be observed at the position around 25 kDa</center>
 +
 
 +
====Differential Scanning Calorimetry (DSC)====
 +
TH is the ability to lower the freezing point of water, which is defined as the gap between incomplete melting point (Th) and freezing point (To). To test the TH ability of TmAFP and SfIBP, BSA was set as the negative control. Each protein was diluted to 50 mM and then was measured by NETZSCH F3 Differential Scanning Calorimetry (please see Experiment for details).
 +
 
 +
As shown in Fig. 3a, Th was determined as -1 ℃, which is lower than the melting temperature. Then To was defined as the beginning temperature of the exothermic peak. The two AFPs, SfIBP and TmAFP, have significant TH activity compared to BSA (Fig. 3b).
 +
 
 +
<center><html><img src="https://static.igem.wiki/teams/4907/wiki/proof-of-concept/poc-fig-3.png" width="400px"></html></center>
 +
<center>Fig. 3 Characterization results of the antifreeze protein. a The DSC scanning curves of BSA. b The DSC scanning curves of SfIBP. c The DSC scanning curves of TmAFP. d The TH of three proteins.</center>
 +
====IRI====
 +
Ice recrystallization inhibition, namely IRI, As shown in Fig. 3a, after a freeze-thaw circle, the recrystallization occurrence temperature of AFPs was also lower than that of BSA too, which indicates the high IRI activity of AFPs.
 +
====Characterization of soil frost resistance====
 +
Equal 50 &mu;M SfIBP solution and Phosphate Buffered Saline (1×PBS) were added in quantitative soil to assay the ability of SfIBP to prevent soil frost.
 +
<center>Fig. 5 Comparison of soil added SfIBP solution and PBS</center>
 +
===Reference===
 +
#T. D. R. Vance, L. A. Graham, P. L. Davies, An ice-binding and tandem beta-sandwich domain-containing protein in <i>Shewanella frigidimarina</i> is a potential new type of ice adhesin. <i>FEBS J.</i> <b>285</b>, 1511-1527 (2018).
 +
#M. Lu et al., Differential Scanning Calorimetric and Circular Dichroistic Studies on Plant Antifreeze Proteins. <i>J. Therm. Anal. Calorim.</i> <b>67</b>, 689-698 (2002).
 +
#M. M. Tomczak, C. B. Marshall, J. A. Gilbert, P. L. Davies, A facile method for determining ice recrystallization inhibition by antifreeze proteins. <i>Biochem. Biophys. Res. Commun.</i> <b>311</b>, 1041-1046 (2003).
  
−
123
 
  
 
<!-- -->
 
<!-- -->

Latest revision as of 14:08, 12 October 2023


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 the freezing point of water, which is defined as the gap between incomplete melting point (Th) and freezing point (To). To test the TH ability of TmAFP and SfIBP, BSA was set as the negative control. Each protein was diluted to 50 mM and then was measured by NETZSCH F3 Differential Scanning Calorimetry (please see Experiment for details).

As shown in Fig. 3a, Th was determined as -1 ℃, which is lower than the melting temperature. Then To was defined as the beginning temperature of the exothermic peak. The two AFPs, SfIBP and TmAFP, have significant TH activity compared to BSA (Fig. 3b).

Fig. 3 Characterization results of the antifreeze protein. a The DSC scanning curves of BSA. b The DSC scanning curves of SfIBP. c The DSC scanning curves of TmAFP. d The TH of three proteins.

IRI

Ice recrystallization inhibition, namely IRI, As shown in Fig. 3a, after a freeze-thaw circle, the recrystallization occurrence temperature of AFPs was also lower than that of BSA too, which indicates the high IRI activity of AFPs.

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]