Difference between revisions of "Part:BBa K3503009"
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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/0/00/T--Puiching_Macau--RFP_blue.png">
 
<div class="legend">Figure 1.Results of inducible expression of RFP-Alpha Casein containing protein validation.
 
<div class="legend">Figure 1.Results of inducible expression of RFP-Alpha Casein containing protein validation.
 
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    <img src="https://2020.igem.org/wiki/images/6/63/T--Puiching_Macau--Alpha_RFP.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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    <img src="https://2020.igem.org/wiki/images/c/c0/T--Puiching_Macau--alpha_RFP_bright.png" style="width:100%">
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    <p style="text-align: center">RFP-Alpha Casein(K3503009)</p>
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<div class="legend">Figure 2. The adhesion level of K3503009(RFP-Alpha casein) by different state of process(non- washing,  after washing with water and soaking.)
 
<div class="legend">Figure 2. The adhesion level of K3503009(RFP-Alpha casein) by different state of process(non- washing,  after washing with water and soaking.)
  

Revision as of 19:56, 26 October 2020


alpha casein- RFP

This is a composite part designed that adding red fluorescence protein(RFP (E1010)). 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 643
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1203
    Illegal AgeI site found at 1315
  • 1000
    COMPATIBLE WITH RFC[1000]


Successful expression of K3503009

In 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: K3507010(CBD-Alpha casein- RFP)
  • Lane8: K3507010(CBD-Alpha casein- RFP) with 0.1mM IPTG induction

The percentage of the protein after processes

RFP-Alpha Casein(K3503009)

Figure 2. The adhesion level of K3503009(RFP-Alpha casein) by different state of process(non- washing, after washing with water and soaking.)
(a)Alpha Casein-RFP(570-620nm)
(b)Alpha Casein-RFP(bright field)

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