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Characterizing Chromoproteins
We encountered problems when measuring the YFP-signal in our C. reinhardtii strains transformed with YFP, even though we were able to detect YFP when examining the cells with the fluorescence microscope. To get a better understanding of the protocols followed to measure absorbance and fluorescence spectra with a plate reader, as an example we conducted some measurements with fluorescent and non-fluorescent chromoproteins expressed in E. coli.
We tested three different chromoproteins from the iGEM Registry of Standard Biological Parts in terms of their absorption spectra (as well as the fluorescence spectrum in case of amajLime):
- amajLime from the coral Anemonia majato BBa_K1033914
- spisPink from the coral Stylophora pistillata BBa_K1033923
- gfasPurple from the coral Galaxea fascicularis BBa_K1033917
Therefore we transformed the pSB1C3 vector with the respective chromoprotein construct (which all carry a promotor and RBS sequence) into E. coli (DH10B Competent Cells). After cultivation we lysed the harvested cells according to our lysis protocol.
With a plate reader (TECAN Plate Reader Infinite 200 Pro) we measured the absorbance (and fluorescence) spectra of the cleared lysate (Figure 3). For the interpretation of the spectra we utilized our lysis buffer as a blank measurement. We obtained characteristic absorbance spectra for all three chromoproteins (Figure 4-6), with the respective absorbance peak equivalent to the values previously described in the literature (Liljeruhm et al. 2018). For the fluorescent chromoprotein amajLime we were able to measure an evident fluorescence spectrum with a clear peak at 493 nm (Figure 7). All spectra are plotted including their respective standard deviations. In Figure 8 the relative absorbance and fluorescence spectra of amajLime are shown to emphasize the Stokes shift of the spectral peaks.
| Table 1. Parameters utilized for the measurement of absorbance and fluorescence spectra | |
|---|---|
| Parameter | Value |
| Number of Samples | 24 |
| Wavelength Step Size | 2 |
| Absorbance Scan: Excitation Wavelength Measurement Range (nm) | [300-800] |
| Fluorescence Scan: Emission Wavelength Measurement Range (nm) | [475-550] |
| Fluorescence Scan: Excitation Wavelength (nm) | 445 |
| Gain (G) | 52 |
| Number of Flashes | 25 |
| Integration Time (µs) | 20 |
| Lag Time (µs) | 0 |
| Settle Time (ms) | 0 |
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Liljeruhm, Josefine et al. “Engineering a palette of eukaryotic chromoproteins for bacterial synthetic biology.” Journal of biological engineering vol. 12:8. 10 May. 2018, doi:10.1186/s13036-018-0100-0