Difference between revisions of "Part:BBa K3503001"
 
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<h2>Sequencing and sequence alignment of K3503001</h2>
 
<h2>Sequencing and sequence alignment of K3503001</h2>
  
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<img style="float:center" src="https://2020.igem.org/wiki/images/5/50/T--Puiching_Macau--K3503001.png">
 
<div class="legend">Figure 1. Representative sequencing results of K3503001
 
<div class="legend">Figure 1. Representative sequencing results of K3503001
  
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For more experimental details of the Western Blot and SDS-PAGE, please see <a href="https://2020.igem.org/Team:PuiChing_Macau/Protocol">protocol</a>.
 
For more experimental details of the Western Blot and SDS-PAGE, please see <a href="https://2020.igem.org/Team:PuiChing_Macau/Protocol">protocol</a>.
  
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    <img src="https://2020.igem.org/wiki/images/d/d5/T--Puiching_Macau--alpha_blue.png" style="width:100%">
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    <p style="text-align: center">(a) Coomassie blue staining</p>
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    <img src="https://2020.igem.org/wiki/images/9/9d/T--Puiching_Macau--Alpha_western.png" style="width:100%">
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    <p style="text-align: center">(b) Western Blot</p>
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<div class="legend">Figure 2. Western Blot results of inducible expression of K3503001, K3503007, K3503008
 
<div class="legend">Figure 2. Western Blot results of inducible expression of K3503001, K3503007, K3503008
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<ul>
 
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<li>Lane1: Bio rad protein dual color ladder;
 
<li>Lane1: Bio rad protein dual color ladder;
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</ul>
 
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<h2>Fire retardant test of K3503001</h2>
 
<h2>Fire retardant test of K3503001</h2>
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<p>The test subject was in the size of bed sheet in 300*130mm and the average Moisture content% is 13.55%-14.37%. As demonstrated on Fig 3, all of our engineered flame retardant displayed a significant improvement compared with the water control.</p>
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<p>The test subject was in the size of bed sheet in 300*130mm and the average Moisture content% is 13.55%-14.37%. As demonstrated on Fig 3, all of our engineered flame retardant displayed an improvement compared with the water control.</p>
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<img src="">
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<img src="https://2020.igem.org/wiki/images/9/9c/T--Puiching_Macau--bedsheet_chart.png">
 
<div class="legend">Figure 3. Vertical Burning Test using bedsheet.     
 
<div class="legend">Figure 3. Vertical Burning Test using bedsheet.     
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<img src="https://2020.igem.org/wiki/images/2/2e/T--Puiching_Macau--wood.png">
 
<div class="legend">Figure 4. Flame retardancy test using wood.
 
<div class="legend">Figure 4. Flame retardancy test using wood.
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<p>As demonstrated in Fig 4, all of our engineered K3503001, K3503007 and K3507008 flame retardants displayed a significant improvement compared with the water control.  </p>
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<p>As demonstrated in Fig 4, all of our engineered K3503001, K3503007 and K3507008 flame retardants displayed an improvement compared with the water control.  </p>
  
 
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Latest revision as of 21:57, 27 October 2020


Alpha-s1-casein-His

The milk protein α-s1-casein from Bos taurus with his tag

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 643
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Sequencing and sequence alignment of K3503001

Figure 1. Representative sequencing results of K3503001

To future confirm that our constructs have the correct sequence, we send our clones (with positive PCR results) out to sequencing.
The sequencing results show that we have successfully assembled target genes K3503001 to the vector (pET-11a).

Successful expression of K3503001

In order to validate the protein expression of K3503001, K3503007 and K3503008, we performed SDS-PAGE (coomassie blue staining) and Western blot analysis. To make K3503001, K3503007 and K3503008 easily detectable via Western Blot, we designed our constructs to include a “His tag” which is targeted by a commercially available antibody. With the SDS-PAGE and western blot (anti-his for his-tagged protein), we found successful expression of K3503001, K3503007 and K3503008.
Before performing the SDS-PAGE, we grew up our engineered E. coli cells containing a plasmid with K3503001, K3503007 and K3503008 under control of the T7 promoter. Cells were induced with0.1mM IPTG until either OD0.4 (wavelength: 600nm) at 30°C. After expression, cells were then lysed according to our protein extraction protocol, and supernatant and pellet were collected for the SDS-PAGE.

For more experimental details of the Western Blot and SDS-PAGE, please see protocol.

(a) Coomassie blue staining

(b) Western Blot

Figure 2. Western Blot results of inducible expression of K3503001, K3503007, K3503008
  • Lane1: Bio rad protein dual color ladder;
  • Lane2: pet11a
  • Lane3: K3503001
  • Lane4:K3503001 with 0.1mM IPTG induction
  • Lane5: K3503007
  • Lane6: K3503007 with 0.1mM IPTG induction
  • Lane7: K3503008
  • Lane8:K3503008 with 0.1mM IPTG induction

Fire retardant test of K3503001

The test subject was in the size of bed sheet in 300*130mm and the average Moisture content% is 13.55%-14.37%. As demonstrated on Fig 3, all of our engineered flame retardant displayed an improvement compared with the water control.

Figure 3. Vertical Burning Test using bedsheet.
Figure 4. Flame retardancy test using wood.

As demonstrated in Fig 4, all of our engineered K3503001, K3503007 and K3507008 flame retardants displayed an improvement compared with the water control.