Difference between revisions of "Part:BBa K3503010"

 
 
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<partinfo>BBa_K3503010 short</partinfo>
 
<partinfo>BBa_K3503010 short</partinfo>
  
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CBD-alpha casein-RFP
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This is a composite part designed that adding red fluorescence protein(E1010). Also adding cellulose binding domain (K1321014)to enhance the fire retardancy of the protein by the adhesion. The protein sequence in this part is based on Alpha s1 casein (K2924026)
  
 
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<partinfo>BBa_K3503010 parameters</partinfo>
 
<partinfo>BBa_K3503010 parameters</partinfo>
 
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<h2>Successful expression of K3503010</h2>
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<p>n order to validate the protein expression of K1608009, K3503010 and K3503011, we performed SDS-PAGE (coomassie blue staining) and Western blot analysis. To make  K1608009, K3503010 and K3503011, 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  K1608009, K3503010 and K3503011.<br>
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Before performing the SDS-PAGE, we grew up our engineered E. coli cells containing a plasmid with K1608009, K3503010 and K3503011 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.
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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>.
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<img src="https://2020.igem.org/wiki/images/0/00/T--Puiching_Macau--RFP_blue.png">
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<div class="legend">Figure 1.Results of inducible expression of RFP-Alpha Casein containing protein validation.
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<ul>
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<li>Lane1: Bio rad protein dual color ladder;
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<li>Lane2: pet11a</li>
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<li>Lane3: K3503009(Alpha casein-RFP)</li>
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<li>Lane4: K3503009(Alpha casein-RFP) with 0.1mM IPTG induction</li>
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<li>Lane5: K3503011(mfp5-Alpha casein-RFP)</li>
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<li>Lane6: K3503011(mfp5-Alpha casein-RFP) with 0.1mM IPTG induction</li>
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<li>Lane7: K3503010(CBD-Alpha casein- RFP)</li>
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<li>Lane8: K3503010(CBD-Alpha casein- RFP) with 0.1mM IPTG induction</li>
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</ul>
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<img src="https://2020.igem.org/wiki/images/f/f1/T--Puiching_Macau--CBD_RFP.png">
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<div class="legend">Figure 2. The adhesion level of K3503010(CBD-Alpha casein-RFP) by different state of process(non- washing,  after washing with water and soaking.)<br><br><br>
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    <img src="https://2020.igem.org/wiki/images/3/30/T--Puiching_Macau--CBD_red.png" style="width:100%">
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    <p style="text-align: center">(a)CBD-Alpha casein-RFP(570-620nm)</p>
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    <img src="https://2020.igem.org/wiki/images/0/02/T--Puiching_Macau--CBD_bright.png" style="width:100%">
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    <p style="text-align: center">(b)CBD-Alpha casein-RFP(bright field)</p>
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  </div>
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<p>Figure 3. The red fluorescence in 570nm-620nm</p>
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Latest revision as of 14:29, 27 October 2020


CBD-alpha casein-RFP

This is a composite part designed that adding red fluorescence protein(E1010). Also adding cellulose binding domain (K1321014)to enhance the fire retardancy of the protein by the adhesion. 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
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1917
    Illegal AgeI site found at 2029
  • 1000
    COMPATIBLE WITH RFC[1000]


Successful expression of K3503010

n order to validate the protein expression of K1608009, K3503010 and K3503011, we performed SDS-PAGE (coomassie blue staining) and Western blot analysis. To make K1608009, K3503010 and K3503011, 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 K1608009, K3503010 and K3503011.
Before performing the SDS-PAGE, we grew up our engineered E. coli cells containing a plasmid with K1608009, K3503010 and K3503011 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.
Figure 1.Results of inducible expression of RFP-Alpha Casein containing protein validation.
  • Lane1: Bio rad protein dual color ladder;
  • Lane2: pet11a
  • Lane3: K3503009(Alpha casein-RFP)
  • Lane4: K3503009(Alpha casein-RFP) with 0.1mM IPTG induction
  • Lane5: K3503011(mfp5-Alpha casein-RFP)
  • Lane6: K3503011(mfp5-Alpha casein-RFP) with 0.1mM IPTG induction
  • Lane7: K3503010(CBD-Alpha casein- RFP)
  • Lane8: K3503010(CBD-Alpha casein- RFP) with 0.1mM IPTG induction
Figure 2. The adhesion level of K3503010(CBD-Alpha casein-RFP) by different state of process(non- washing, after washing with water and soaking.)


(a)CBD-Alpha casein-RFP(570-620nm)

(b)CBD-Alpha casein-RFP(bright field)

Figure 3. The red fluorescence in 570nm-620nm