Part:BBa_K1890010

mCerulean with strong consitutive promoter and RBS

Indroduction

mCerulean is a cyan fluorescent protein derived from ECFP. It presents a series of mutations that increase its extinction coefficient, quantum yield and fluorescence lifetime [1]. It has a major emission peak at 475 nm and a minor one at 503 nm. Its excitation peaks are at 433 nm and 445 nm. We expressed it under the control of the strong constitutive promoter BBa_J23100 and strong RBS BBa_B0030.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Characterization

This part was expressed in E. coli strain BL21. After transformation the cells adapted a bright yellow colour (Figure 1).

Figure 1. E. coli expressing mCerulean (left) and not expressing mCerulean (right) in eM9 medium. The mCerulean expressing cultures show a bright yellow colour.

After transformation, the following experiments were performed to verify the fluorescence:

• Measurement of excitation and emission spectrum.
• Viability assay.
• Fluorescence imaging.

Excitation and emission

In order to make sure that mCerulean was expressed and functioning properly, the excitation and emission spectra were recorded in a plate reader (Figure 2). The cells were grown in LB medium and washed in PBS. For measurement of the excitation and emission spectra, an emission wavelength of 475 and an excitation wavelength of 433 nm were used.

Figure 2. Excitation and emission spectrum of mCerulean.

Both spectra are as expected according to Rizzo et al. (2004), from which we can conclude that mCerulean is expressed and functioning properly.

Viability assay

To investigate whether the constitutive expression of this fluorophore affected the cell growth, we performed a growth study. Cell expressing mCerulean were compared with cells expressing GFP under the same promoter and RBS (BBa_K1890020). An overnight culture in eM9 medium was inoculated in fresh eM9 to an OD600 of 0.1 in a 96 well plate. The emission at 522 nm was measured every 15 minutes. Measurements were done in quadruplicate with pure eM9 as a blank. Figure 3 shows the 24 hour measurement of optical density and fluorescence intensity.

Figure 3. Growth and fluorescence intensity of E. coli expressing GFP (top) or mCerulean (bottom).

Cells expressing mCerulean seem to be having a longer lag phase before exponential growth starts than the ones expressing GFP. Also, they reach a lower final OD600 than the ones expressing GFP. This phenomenon was also observed while preparing overnight cultures. Cultures expressing mCerulean typically took one day longer to reach the same OD600 as the ones expressing other plasmids. Concluding, constitutive expression of mCerulean might be slightly harmful for the cell. This did not result in major problems, as the cells could still be cultivated successfully.

Fluorescence imaging

The cells were imaged in a setup especially built to image these fluorescent cells (see our <a href="http://2016.igem.org/Team:TU_Delft/Hardware"wiki</a>). They were excited with a laser at a wavelength of 405 nm and an intensity of 0.5 mW.

Figure 4. mCerulean expressing E. coli excited with a laser at a wavelength of 405 nm.

References

[1] Rizzo, M. A., Springer, G. H., Granada, B. & Piston, D. W. An improved cyan fluorescent protein variant useful for FRET. Nat. Biotechnol. 22, 445–449 (2004).