Part:BBa_K1890011

mVenus with strong consitutive promoter and RBS

Indroduction

mVenus is a variant of yellow fluorescent protein (YFP) which contains several mutations that make it more stable and provide an improved speed and efficiency of maturation. It has its main emission peak at 530 nm and main excitation peak at 515 nm [1]. We expressed it under the control of the strong constitutive promoter BBa_J23100 and 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 following experiments were performed to verify the fluorescence:

• Measurement of emission spectrum.
• Viability assay.

Emission

In order to make sure that mVenus was expressed and functioning properly, the emission spectra was recorded in a plate reader (Figure 1). The cells were grown in LB medium and washed in PBS. For measurement an excitation wavelength of 510 nm was used, rather than 515 nm, in order to have the excitation and emission less close together.

Figure 1. Emission spectrum of mVenus.

Because of the fact that the excitation and emission wavelength are so close together, it was not possible to measure emissions under 530 nm without being within the bandwidth of the excitation source. Apart from that, the spectrum is as expected according to Nagai et al. (2002), from which we can conclude that mVenus is expressed and functioning properly.

References

[1] Nagai, T. et al. A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat. Biotechnol. 20, 87–90 (2002).