Difference between revisions of "Part:BBa K3503008"
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     <img src="https://2020.igem.org/wiki/images/d/d5/T--Puiching_Macau--alpha_blue.png" style="width:100%">
 
     <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">The percentage of the protein after processes</p>
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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/2/2f/T--Puiching_Macau--CBD_alpha_western.png" style="width:100%">
 
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     <p style="text-align: center">RFP-Alpha Casein(K3503009)</p>
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     <p style="text-align: center">(b) Western Blot</p>
 
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Revision as of 20:17, 26 October 2020


CBD-alpha casein-His

This is a composite part designed to improve the fire retardancy with mussel cellulose binding domain (K1321014). The protein sequence in this part is based on Alpha s1 casein (K2924026) 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 1357
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Successful expression of K3503008

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 1. Western Blot results of inducible expression of K3503001, K3503007, K3503008
(a)
  • 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 K3503008

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 2, all of our engineered flame retardant displayed a significant improvement compared with the water control.

Figure 2. Vertical Burning Test using bedsheet.
Figure 3. Flame retardancy test using wood.

As demonstrated in Fig 3, all of our engineered K3503001, K3503007 and K3507008 flame retardants displayed a significant improvement compared with the water control.