Difference between revisions of "Part:BBa K1976002"

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             In some cases a fast signaling reporter, as well as a fast decay of the said reporter is necessary to create a specific genetic circuit. One of the best fitting reporters might be the <i>E.coli</i> optimized version of mVenus. With an average maturation time of 40 min in vitro it is faster than GFP. In order to prevent a persistent fluorescence after the expression of the reporter stopped, mVenus is expressed with a LVA degradation tag to decrease the protein half-life. Another positive aspect of mVenus is the lowered sensitivity towards pH and chloride ion concentration, one of the drawbacks of wild-type GFP. The lack of disulfide bonds enables fluorescence under reductive conditions. Moreover, the reporter is not regulated by any proteins, cofactors or substrates. Therefore mVenus does not only expand the spectrum of fluorescence proteins in the registry, it also is a good alternative for various genetic circuits<sup>1,2</sup>.
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             In some cases a fast signaling reporter, as well as a fast decay of the said reporter is necessary to create a specific genetic circuit. One of the best fitting reporters might be the <i>E.coli</i> optimized version of mVenus. With an average maturation time of 40 min in vitro it is faster than GFP. In order to prevent a persistent fluorescence after the expression of the reporter stopped, mVenus is expressed with a LVA degradation tag to decrease the protein half-life. Another positive aspect of mVenus is the lowered sensitivity towards pH and chloride ion concentration, one of the drawbacks of wild-type GFP. The lack of disulfide bonds enables fluorescence under reductive conditions. Moreover, the reporter is not regulated by any proteins, cofactors or substrates. Therefore mVenus does not only expand the spectrum of fluorescence proteins in the registry, it also is a good alternative for various genetic circuits<sup>1,2,3</sup>.
  
 
      
 
      
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             <b>Figure 1: </b>Excitation and emission spectrum of mVenus.The fluorescent
 
             <b>Figure 1: </b>Excitation and emission spectrum of mVenus.The fluorescent
 
protein excitation intensity at 280 nm is thought to be due to fluorescence resonance energy transfer from aromatic
 
protein excitation intensity at 280 nm is thought to be due to fluorescence resonance energy transfer from aromatic
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amino acids to the chromophore<sup>2</sup>.
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amino acids to the chromophore<sup>4</sup>.
 
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As seen in Figure2,the maximum of exication is at 515 nm and the maximum of emission at 528 nm.
 
As seen in Figure2,the maximum of exication is at 515 nm and the maximum of emission at 528 nm.
 
mVenus has a brightness of 15, which is defined as the roduct of the extinction coefficient and the quantum yield at pH 7.4 under ideal maturation conditions. Its photostabillity is 15 s, where the photostabillity is defined as the time for bleaching from an initial emission rate of 1,000 photons/s
 
mVenus has a brightness of 15, which is defined as the roduct of the extinction coefficient and the quantum yield at pH 7.4 under ideal maturation conditions. Its photostabillity is 15 s, where the photostabillity is defined as the time for bleaching from an initial emission rate of 1,000 photons/s
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down to 500 photons/s. its pka is located at 6.0. it is a weak dimer<sup>3</sup>.
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down to 500 photons/s. its pka is located at 6.0. it is a weak dimer<sup>5</sup>.
  
 
<h2>Sequence and Features</h2>
 
<h2>Sequence and Features</h2>
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<b>References</b>
 
<b>References</b>
 
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[1] Agata Rekas, Jean-René Alattia, Takeharu Nagai, Atsushi Miyawaki and Mitsuhiko Ikura, Crystal Structure of Venus, a Yellow Fluorescent Protein with Improved Maturation and Reduced Environmental Sensitivity, J. Biol. Chem., vol 277, pp. 50573-50578, 2002
 
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2. Pabak Sarkar, Srinagesh V. Koushik et al. (2009) Photophysical Properties of Cerulean and Venus Fluorescent
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[2] Nagai Takeharu, Ibata Keiji,Park Eun Sun, Kubota Mie, Mikoshiba Katsuhiko, A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications, Nature Biotechnology, vol. 20, pp. 1585-1588, 2001
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[3] Sarkar, Pabak Koushik, Srinagesh V Vogel, Steven S Gryczynski, Ignacy Gryczynski, Zygmunt, Photophysical Properties of Cerulean and Venus Fluorescent Proteins, Journal of Biomedical Optics, vol 14, pp. 1-25, 2009
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[4] Pabak Sarkar, Srinagesh V. Koushik et al. (2009) Photophysical Properties of Cerulean and Venus Fluorescent
 
Proteins - J Biomed Opt. 2009 ; 14(3): 034047. doi:10.1117/1.3156842
 
Proteins - J Biomed Opt. 2009 ; 14(3): 034047. doi:10.1117/1.3156842
 
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3. Nathan C Shaner1, Paul A Steinbach & Roger Y Tsien (2005) A guide to choosing fluorescent proteins - Nature Methods
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[5] Nathan C Shaner1, Paul A Steinbach & Roger Y Tsien (2005) A guide to choosing fluorescent proteins - Nature Methods
 
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Revision as of 22:22, 21 October 2016

mVenus with a LVA degradation tag

This part contains an E. coli optimized coding sequence of the yellow fluorescent protein mVenus with an associated LVA degradation tag.

Usage

In some cases a fast signaling reporter, as well as a fast decay of the said reporter is necessary to create a specific genetic circuit. One of the best fitting reporters might be the E.coli optimized version of mVenus. With an average maturation time of 40 min in vitro it is faster than GFP. In order to prevent a persistent fluorescence after the expression of the reporter stopped, mVenus is expressed with a LVA degradation tag to decrease the protein half-life. Another positive aspect of mVenus is the lowered sensitivity towards pH and chloride ion concentration, one of the drawbacks of wild-type GFP. The lack of disulfide bonds enables fluorescence under reductive conditions. Moreover, the reporter is not regulated by any proteins, cofactors or substrates. Therefore mVenus does not only expand the spectrum of fluorescence proteins in the registry, it also is a good alternative for various genetic circuits1,2,3.

Characteristica

Below are some characteristica of mVenus listed.

Figure 1: Excitation and emission spectrum of mVenus.The fluorescent protein excitation intensity at 280 nm is thought to be due to fluorescence resonance energy transfer from aromatic amino acids to the chromophore4.


As seen in Figure2,the maximum of exication is at 515 nm and the maximum of emission at 528 nm. mVenus has a brightness of 15, which is defined as the roduct of the extinction coefficient and the quantum yield at pH 7.4 under ideal maturation conditions. Its photostabillity is 15 s, where the photostabillity is defined as the time for bleaching from an initial emission rate of 1,000 photons/s down to 500 photons/s. its pka is located at 6.0. it is a weak dimer5.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


References
[1] Agata Rekas, Jean-René Alattia, Takeharu Nagai, Atsushi Miyawaki and Mitsuhiko Ikura, Crystal Structure of Venus, a Yellow Fluorescent Protein with Improved Maturation and Reduced Environmental Sensitivity, J. Biol. Chem., vol 277, pp. 50573-50578, 2002
[2] Nagai Takeharu, Ibata Keiji,Park Eun Sun, Kubota Mie, Mikoshiba Katsuhiko, A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications, Nature Biotechnology, vol. 20, pp. 1585-1588, 2001
[3] Sarkar, Pabak Koushik, Srinagesh V Vogel, Steven S Gryczynski, Ignacy Gryczynski, Zygmunt, Photophysical Properties of Cerulean and Venus Fluorescent Proteins, Journal of Biomedical Optics, vol 14, pp. 1-25, 2009
[4] Pabak Sarkar, Srinagesh V. Koushik et al. (2009) Photophysical Properties of Cerulean and Venus Fluorescent Proteins - J Biomed Opt. 2009 ; 14(3): 034047. doi:10.1117/1.3156842
[5] Nathan C Shaner1, Paul A Steinbach & Roger Y Tsien (2005) A guide to choosing fluorescent proteins - Nature Methods