Difference between revisions of "Part:BBa K1141002"
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<p align="center"><img src="https://static.igem.org/mediawiki/2013/0/01/KR_Biobrick_gel.png" alt="KR SDS-PAGE purification gel" width="1000px"></p>
 
<p align="center"><img src="https://static.igem.org/mediawiki/2013/0/01/KR_Biobrick_gel.png" alt="KR SDS-PAGE purification gel" width="1000px"></p>
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                                         <p align="center" id="legend">Figure 1: Left: colonies obtained with BBa_K1141002 on an LB-Agar plate. Right: An agarose gel showing a digestion experiment with BBa_K1141002. In order from left to right:<ol><br>
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                                         <p align="center" id="legend">Figure 1: Left: colonies obtained with BBa_K1141002 on an LB-Agar plate. Right: An agarose gel showing a digestion experiment with BBa_K1141002.</p>
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<p>In order from left to right:<br><br>
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<ol>
 
<li>MassRuler Molecular Weight Ladder</li>
 
<li>MassRuler Molecular Weight Ladder</li>
 
<li>pSB1C3 digested with Eco RI and Pst I</li>
 
<li>pSB1C3 digested with Eco RI and Pst I</li>

Revision as of 18:51, 4 October 2013

Plac-RBS-KillerRed (IPTG-inducible)

KillerRed is a red fluorescent protein which produces ROS under green illumination (roughly 500-600 nm, absorption peak at 535 nm). This biobrick allows production of KillerRed under the control of the PLAC promoter (In this construction, actually a T5 phage promoter preceded by two lac operators for strong repression in presence of LacI).With this biobrick the production of KillerRed is IPTG-inducible. We used this part to trigger cell death in response to light illumination for controlling cell density in the Talk'E.coli project of Grenoble-EMSE-LSU 2013. KillerRed is a red fluorescent protein with Absorption in the green portion and emission in the red portion of the visible spectrum (figure 1):

Killer Red absorption-emission spectra

Figure 1:The KillerRed protein absorption (left peak) and emission (right peak) spectra
Source: Detailed KillerRed description from Evrogen

Note that the information characterising this part was made with BBa_K1141001, which has the same sequence except for an additional Eco RI site between the Promoter and RBS. The vector for BBa_K1141001 is a high copy plasmid like pSB1C3 with ac col1 as duplication origin. KillerRed made by this biobrick dimerizes and isn't suitable for protein fusions. We verified the presence of the correct sequence inside pSB1C3 by qualitative analysis of colonies (the red colour shows function of the biobrick in producing a red fluorescent protein) and by digestion on agarose gel:

KR SDS-PAGE purification gel

Figure 1: Left: colonies obtained with BBa_K1141002 on an LB-Agar plate. Right: An agarose gel showing a digestion experiment with BBa_K1141002.

In order from left to right:

  1. MassRuler Molecular Weight Ladder
  2. pSB1C3 digested with Eco RI and Pst I

  3. Below is a SDS-PAGE gel showing purification of the protein, visible near the 31 kDa mark on the molecular weight ladder (figure 2):

    KR SDS-PAGE purification gel

    Figure 2:KillerRed as can be obtained on an SDS-PAGE gel after purification. The protein stain can be clearly seen at the 31 kDa mark.

    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 133
    • 23
      COMPATIBLE WITH RFC[23]
    • 25
      COMPATIBLE WITH RFC[25]
    • 1000
      INCOMPATIBLE WITH RFC[1000]
      Illegal BsaI.rc site found at 289
      Illegal BsaI.rc site found at 580