Difference between revisions of "Part:BBa K3503011"
 
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<partinfo>BBa_K3503011 parameters</partinfo>
 
<partinfo>BBa_K3503011 parameters</partinfo>
 
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<h2>Successful expression of K3503011</h2>
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<p>In order to validate the protein expression of K3503009, K3503010 and K3503011, we performed SDS-PAGE (coomassie blue staining) and Western blot analysis. To make  K3503009, 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  K3503009, K3503010 and K3503011.
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Before performing the SDS-PAGE, we grew up our engineered E. coli cells containing a plasmid with K3503009, 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/0/04/T--Puiching_Macau--mfp5_RFP.png">
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<div class="legend">Figure 2. The adhesion level of K3503011(mfp5-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/e/ea/T--Puiching_Macau--mfp5_red.png" style="width:100%">
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    <p style="text-align: center">(a)mfp5-Alpha casein-RFP(570-620nm)</p>
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    <img src="https://2020.igem.org/wiki/images/0/0b/T--Puiching_Macau--mfp5_bright.png" style="width:100%">
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    <p style="text-align: center">(b)mfp5-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 14:28, 27 October 2020


mfp5-alpha casein-RFP

This is a composite part designed that adding red fluorescence protein(E1010). Also, we added the mussel adhesion protein (K308902) to enhance the adhesion of the protein to have a better fire-defending. 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 727
    Illegal BamHI site found at 1525
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 2085
    Illegal AgeI site found at 2197
  • 1000
    COMPATIBLE WITH RFC[1000]


Successful expression of K3503011

In order to validate the protein expression of K3503009, K3503010 and K3503011, we performed SDS-PAGE (coomassie blue staining) and Western blot analysis. To make K3503009, 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 K3503009, K3503010 and K3503011. Before performing the SDS-PAGE, we grew up our engineered E. coli cells containing a plasmid with K3503009, 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 K3503011(mfp5-Alpha casein-RFP) by different state of process(non- washing, after washing with water and soaking.)


(a)mfp5-Alpha casein-RFP(570-620nm)

(b)mfp5-Alpha casein-RFP(bright field)

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