Difference between revisions of "Part:BBa K1141002"
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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. | 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: | ||
+ | |||
+ | <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" 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> | ||
+ | <li>MassRuler Molecular Weight Ladder</li> | ||
+ | <li>pSB1C3 digested with Eco RI and Pst I</li> | ||
+ | <li></li> | ||
+ | </p> | ||
Below is a SDS-PAGE gel showing purification of the protein, visible near the 31 kDa mark on the molecular weight ladder (figure 2): | Below is a SDS-PAGE gel showing purification of the protein, visible near the 31 kDa mark on the molecular weight ladder (figure 2): | ||
<p align="center"><img src="https://static.igem.org/mediawiki/2013/6/6f/KR_SDS_Purification.JPG" alt="KR SDS-PAGE purification gel" width="500px"></p> | <p align="center"><img src="https://static.igem.org/mediawiki/2013/6/6f/KR_SDS_Purification.JPG" alt="KR SDS-PAGE purification gel" width="500px"></p> | ||
− | <p align="center" id="legend">Figure | + | <p align="center" id="legend">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.<br> |
</p> | </p> | ||
Revision as of 18:50, 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):
Figure 1:The KillerRed protein absorption (left peak) and emission (right peak) spectra
Source: Detailed KillerRed description from Evrogen
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:
- MassRuler Molecular Weight Ladder
- pSB1C3 digested with Eco RI and Pst I Below is a SDS-PAGE gel showing purification of the protein, visible near the 31 kDa mark on the molecular weight ladder (figure 2):
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 133
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 289
Illegal BsaI.rc site found at 580
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