Part:BBa_K1141002:Experience

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 Cell Killing:

On the graph above, an experiment with KillerRed using mCherry as negative control was performed. Cells expressing either KillerRed or mCherry with BBa_K1141001 (same sequence as BBa_K1141002) were inoculated into M9 minimal medium (recipe here). After a period of growth in darkness,

Bacterial Growth Regulation with light intensity:

The curves above show the evolution of culture OD610 and KillerRed fluorescence (relative light units) with BBa_K1141001 at different light intensities. The experiment shows that the cell killing and photobleaching effects are a function of light intensity: the higher the light intensity, the stronger the effects. Note that photobleaching (destruction of KillerRed's chromophore, making the protein colourless and non-toxic) is a phenomenon that is present in all fluorescent proteins but that KillerRed is particularly susceptible to photobleaching.

Finally, we can show that cells survive after illumination while keeping BBa_K1141001.

===Usage of BBa_K1141002 Fluorescent Protein Characteristics:

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 detailed description. 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):

Correct IPTG concentration

Using Bba_K1141002 with induction of PLac at saturating concentrations of IPTG (1 mM) has a severe effect on the gowth of cells, which is significantly slowed. During the 2013 Grenoble-EMSE-LSU team's project, we determined an optimal IPTG concentration to use for good KillerRed expression while avoiding any effect on the growth of cells, while the culture is kept in the dark.
Below is a comparison of growth curves obtained with BBa_K1141000 (mCherry negative control) and BBa_K1141001 (same sequence as for BBa_K1141002, but in the commercial pQE30 plasmid) at different IPTG concentrations:



Note the lack of effect on cell growth for all IPTG concentrations with BBa_K1141000 (PLac-RBS-mCherry). This is expected behaviour for proteins that are non toxic to E. coli. High IPTG concentrations with cells containing BBa_K1141001 will significantly slow their growth, which can be explained by KillerRed's intrinsic cytotoxicity, even in darkness. It can be obsrved that the best results obtained for cell growth and KillerRed expression with BBa_K1141001 were with a concentration of IPTG=0.05 mM. This was the optimal IPTG concentration we used in further experiments with BBa_K1141001.

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