Difference between revisions of "Part:BBa K1141002:Experience"

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===Applications of BBa_K1141002===
 
===Applications of BBa_K1141002===
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Bacterial Growth Regulation:
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===Usage of BBa_K1141002
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Fluorescent Protein Characteristics:<br><br>
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<p align="center"><img src="https://static.igem.org/mediawiki/2013/7/7f/KillerRed_spectra2.png" alt="Killer Red absorption-emission spectra" width="500px"></p>
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KillerRed is a type of Red Fluorescent Protein, with the absorption and emission spectra shown above. Left peak is absorption, right peak is emission. The optical properties can be obtained from EVROGEN's <a href="http://www.evrogen.com/products/KillerRed/KillerRed_Detailed_description.shtml">detailed description</a>. Credits to EVROGEN for the spectra.<br>
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It is advised to illuminate the protein with a large portion of the green spectrum, as illumination with green laser light at 535 nm has been reported to us as ineffective. In our experiments, we have illuminated it with a white LED light (INSERT REFERENCES HERE), and our observations showed the protein to be active when illuminated in this manner.
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Expression strain:
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During our experiments with KillerRed we used QIAGEN's M15 strain of <em>E. coli</em>. This strain is Thy- (no thymine production, meaning no vitamin B production). It needs to be grown in rich media or in minimal media with vitamin B as a supplement. Our experiments used M9 minimal media with added vitamin B (See Grenoble-EMSE-LSU 2013 wiki for exact media composition).<br>
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M15 cells contain the PLAC repressor plasmid pREP4 (Kanamycin resistance) which provides efficient repression of the notably leaky PLAC used in pQE30. This PLAC can be found in BBa_K1141001 and BBa_K1141002. M15 cells with the pREP4 plasmid and BBa_K1141001 or BBa_K1141002 do not express KillerRed until introduction of IPTG into the culture. The following experiment shows production of KillerRed as a function of IPTG concentration (millimolar):<br><br>
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<p align="center"><img src="https://static.igem.org/mediawiki/2013/f/fa/Strain_choice.png" alt="Bacterial growth curves for KillerRed-expressing M15 and BW25113 (wild-type control) cells" width="500px"></p>
  
  

Revision as of 08:43, 2 October 2013

Here we have posted our experimental results regarding applications of KillerRed: its characterization and our choice of expressing strains for the protein.

Applications of BBa_K1141002

Bacterial Growth Regulation:

===Usage of BBa_K1141002

Fluorescent Protein Characteristics:

<img src="KillerRed_spectra2.png" alt="Killer Red absorption-emission spectra" width="500px">

KillerRed is a type of Red Fluorescent Protein, with the absorption and emission spectra shown above. Left peak is absorption, right peak is emission. The optical properties can be obtained from EVROGEN's <a href="http://www.evrogen.com/products/KillerRed/KillerRed_Detailed_description.shtml">detailed description</a>. Credits to EVROGEN for the spectra.
It is advised to illuminate the protein with a large portion of the green spectrum, as illumination with green laser light at 535 nm has been reported to us as ineffective. In our experiments, we have illuminated it with a white LED light (INSERT REFERENCES HERE), and our observations showed the protein to be active when illuminated in this manner.

Expression strain: During our experiments with KillerRed we used QIAGEN's M15 strain of E. coli. This strain is Thy- (no thymine production, meaning no vitamin B production). It needs to be grown in rich media or in minimal media with vitamin B as a supplement. Our experiments used M9 minimal media with added vitamin B (See Grenoble-EMSE-LSU 2013 wiki for exact media composition).
M15 cells contain the PLAC repressor plasmid pREP4 (Kanamycin resistance) which provides efficient repression of the notably leaky PLAC used in pQE30. This PLAC can be found in BBa_K1141001 and BBa_K1141002. M15 cells with the pREP4 plasmid and BBa_K1141001 or BBa_K1141002 do not express KillerRed until introduction of IPTG into the culture. The following experiment shows production of KillerRed as a function of IPTG concentration (millimolar):

<img src="Strain_choice.png" alt="Bacterial growth curves for KillerRed-expressing M15 and BW25113 (wild-type control) cells" width="500px">


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