Difference between revisions of "Part:BBa K4907001"

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The plasmids verified by sequencing was successfully transformed into <i>E. coli</i> BL21 Star(DE3). After being cultivated and induced by 0.7 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 (12.2 kDa) can be observed at the position around 10 kDa on the purified protein lanes (FR), although displayed with many other protein bands together.
 
The plasmids verified by sequencing was successfully transformed into <i>E. coli</i> BL21 Star(DE3). After being cultivated and induced by 0.7 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 (12.2 kDa) can be observed at the position around 10 kDa on the purified protein lanes (FR), although displayed with many other protein bands together.
  
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<center>Fig. 2 SDS-PAGE analysis of his-SfIBP protein. Target bands (12.2 kDa) can be observed at the position around 10 kDa.</center>
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<center>Fig. 2 SDS-PAGE analysis of his-SfIBP protein. Target bands (12.2 kDa) can be observed at the position around 10 kDa</center>
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====Differential Scanning Calorimetry (DSC)====
 
====Differential Scanning Calorimetry (DSC)====
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TH is the ability to lower freezing point (T<sub>f</sub>) of water, which is defined as the gap between melting point (T<sub>m</sub>) and freezing point (T<sub>f</sub>). As Fig. 3 shows, both T<sub>m</sub> and T<sub>f</sub> of 50 mu;M TmAFP solution were measured by DSC (2), then we determined the TH activity of TmAFP.
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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).
  
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<center>Fig. 3 DSC curve</center>
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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).
  
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<center><html><img src="https://static.igem.wiki/teams/4907/wiki/proof-of-concept/poc-fig-3.png" width="400px"></html></center>
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<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====
 
====IRI====
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The least concentration of AFPs presenting IRI activity is defined as the end point of AFPs. It represents the IRI activity of AFPs, and can be detected by [https://2023.igem.wiki/xmu-china/results the capillary methods] (3).
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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.
  
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<center>Fig. 4 IRI assay of TmAFP by the capillary method.</center>
 
  
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1. Antifreeze at soil
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===Antifreeze at soil===
 
Equal 50 &micro;M TmAFP solution and Phosphate Buffered Saline (1 ×PBS) were added in quantitative soil to assay the ability of TmAFP to prevent soil frost.
 
Equal 50 &micro;M TmAFP solution and Phosphate Buffered Saline (1 ×PBS) were added in quantitative soil to assay the ability of TmAFP to prevent soil frost.
  

Latest revision as of 14:10, 12 October 2023


tmafp-his tag

Biology

TmAFP

TmAFP, found from Tenebrio molitor, is one of antifreeze proteins (AFPs) (1). It is capable to prevent liquid freezing and inhibit ice growth, which is defined as Thermal Hysteresis (TH) and ice recrystallization inhibition (IRI) respectively. Notably, TmAFP possess highest TH activity between all AFPs that have been found.

Usage

In order to measure TH and IRI activity of TmAFP, and finally verify if it is capable to solve soil frost problem, a His-tag (6×his) was added to the C-terminal of TmAFP 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 Star(DE3), then the positive transforms were selected by kanamycin and confirmed by colony PCR. Target bands (465 bp) can be observed at the position around 500 bp. (Fig. 1)

Fig. 1 colony PCR of BBa_K4907001_pET-28a(+) in E.coli BL21 Star(DE3)

SDS-PAGE

The plasmids verified by sequencing was successfully transformed into E. coli BL21 Star(DE3). After being cultivated and induced by 0.7 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 (12.2 kDa) can be observed at the position around 10 kDa on the purified protein lanes (FR), although displayed with many other protein bands together.

Fig. 2 SDS-PAGE analysis of his-SfIBP protein. Target bands (12.2 kDa) can be observed at the position around 10 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.


Antifreeze at soil

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

Fig. 5 Comparison of soil added TmAFP solution and PBS

Reference

  1. M. U. Sankar, B. Sanjoy, Elucidating the Sluggish Water Dynamics at the Ice-Binding Surface of the Hyperactive Tenebrio molitor Antifreeze Protein. J. Phys. Chem. B 127, 121-132 (2023).
  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 202
  • 1000
    COMPATIBLE WITH RFC[1000]