Difference between revisions of "Part:BBa K1890010"
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<h2>Indroduction</h2>
 
<h2>Indroduction</h2>
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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]. We expressed it under the control of the strong constitutive promoter <partinfo>BBa_J23100</partinfo> and strong RBS <partinfo>BBa_B0030</partinfo>.
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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 <partinfo>BBa_J23100</partinfo> and strong RBS <partinfo>BBa_B0030</partinfo>.
  
 
<h2><span class='h3bb'>Sequence and Features</span></h2>
 
<h2><span class='h3bb'>Sequence and Features</span></h2>
 
<partinfo>BBa_K1890010 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K1890010 SequenceAndFeatures</partinfo>
  
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<h2>References</h2>
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<h2>Characterization</h2>
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[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).
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This part was expressed in <i>E. coli</i> strain BL21. After transformation the cells adapted a bright yellow colour (Figure 1).
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<html>
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    <figure>
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    <center><img src="https://static.igem.org/mediawiki/2016/7/79/T--TU_Delft--mCerulean_tube.jpg" alt="mCerulean">
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        <figcaption><b>Figure 1.</b> <i>E. coli</i> expressing mCerulean (left) and not expressing mCerulean (right). The mCerulean expressing cultures show a bright yellow colour.</figcaption></center>
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</figure>
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</html>
  
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After transformation, the following experiments were performed to verify the fluorescence:
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* Measurement of excitation and emission spectrum.
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* Viability assay.
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* Fluorescence imaging.
  
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<h3>Excitation and emission</h3>
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===Functional Parameters===
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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 was used.
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<partinfo>BBa_K1890010 parameters</partinfo>
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<html>
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<!-- -->
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    <figure>
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    <center><img src="https://static.igem.org/mediawiki/2016/9/94/T--TU_Delft--Spectrum_mCerulean.png" alt="mCerulean">
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        <figcaption><b>Figure 2.</b> Excitation and emission spectrum of mCerulean.</figcaption></center>
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</figure>
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</html>
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Both spectra are as expected according to Rizzo <i>et al.</i> (2004), from which we can conclude that mCerulean is expressed and functioning properly.
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<h2>References</h2>
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[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).

Revision as of 14:18, 16 October 2016


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).

mCerulean
Figure 1. E. coli expressing mCerulean (left) and not expressing mCerulean (right). 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 was used.

mCerulean
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.

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).