Part:BBa_K256033:Experience

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Applications of BBa_K256033

Created in Tsien lab, Infrared fluorescent protein is a monomeric protein engineered from a bacterial phytochrome of the species Deinococcus radiodurans. This bacteriophytochrome incorporates biliverdin IXα (BV) as its chromophore. Biliverdin IXα is a natural product of heme catabolism by the enzyme heme oxygenase-1 (HO-1), it is essential to stabilize IFP. By coexpressing both HO1 and IFP, infrared fluorescent signal can be produced for in vivo imaging.

Characterization Data

The resuspended cell cultures in 2mL of LB medium were irradiated with a 660 nm laser diode for excitation. Ocean Optics USB4000 Portable Spectrometer was used to detect the infrared emission of the cell cultures at circa 708nm. Our K256033 emission peak (purple) has an intensity value of 3533.37 counts and closely follows the shape profile of our positive control, IFP1.4 from the Tsien's Lab (green). Dark spectrum refers to the spectrum obtained when there is no light source while blank spectrum is our reference spectrum using competent cells (E. coli strain K12)as negative control.

Temperature Dependence

Fig: Infrared Emission Spectrum of BBa_K256033 as a function of temperature. Plaque Out! imaging test construct (Part:BBa_K256033) was incubated for 14 hours at 25oC, 30oC and 37oC. The cell cultures were centrifuged at 4000 rpm for 10 minutes and subsequently resuspended in 2mL of LB medium for infrared fluorescence detection by Ocean Optics USB4000 Portable Spectrometer. Cell culture incubated at 37oC gives the strongest intensity level of 3597.31 counts at 708.10 nm, followed by that of 30oC which gives 2030.83 counts and the cell culture incubated at 25oC has the lowest intensity of 1521.94 counts. This result characterizes BBa_K256033, establishing a relationship of temperature dependence for the intensity of infrared fluorescence emission.

Concentration Dependence

Fig: Infrared Emission Spectrum of BBa_K256033 as a function of concentration. Incubation temperature was kept constant at 37oC. 14-hour cell cultures were centrifuged at 4000 rpm for 10 minutes. Cell pellets from 10mL, 20mL and 30mL were resuspended with 2 mL of LB medium which gives 5X, 10X and 15X final concentrations respectively. The intensity level of infrared fluorescence emission from the 15X final concentration is approximately doubles that of the 5X final concentration.

Fig: Temperature and Concentration Dependence of BBa_K256033. Increasing trend is observed from 25oC to 37oC of incubation temperature and from 5X to 15X final concentration of resuspended cell pellets. This result shows the influence of temperature on cell growth as well as influence of final concentration of cells on intensity level. BBa_K256033 can therefore yield highest infrared fluorescence intensity when it is concentrated to 15X final concentration and incubated for growth at 37oC as compared to other conditions.

pH Dependence

Fig: Infrared fluorescence intensity of BBa_K256033 as a function of pH. Cell cultures were incubated at 37oC for 14 hours before centrifugation at 4000 rpm for 10 minutes. To vary the pH of the resuspension buffer, Phosphate Buffered Saline (PBS) was prepared for pH 4.0, pH 5.3, pH 7.0 and pH 10.0. For each pH, cell pellets of 40mL were resuspended to 8mL of PBS solution to give a 5X final concentration. Sonication and subsequently centrifugation of the resuspended cell cultures were performed. 2mL of the each supernatant of the pH was transferred into fluorimetric 4-clear-side cuvette for infrared fluorescence intensity reading with Ocean Optics USB4000 Portable Spectrometer. At pH 4.0, the averaged intensity at 708.10 nm is 108.17 counts, almost comparable to the blank spectrum (competent cells). The plot shows a maximum intensity value of fluorescence at pH 7.0 as opposed to pH 4.0, pH 5.3 and pH 10.0.

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