Difference between revisions of "Part:BBa K1976002"
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===<h2>Characteristics</h2>=== | ===<h2>Characteristics</h2>=== | ||
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<b>mVenus</b> | <b>mVenus</b> | ||
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The weak dimeric flourophor mVenus, with an atomic mass of approximately 27 kDA and a pkA of 6.0, has its absorption maximum at 512 nm and its emission maximum at 528 nm. With a photostabillity of 15, it is one of the fastest decaying flourorphores. The photostabillity is defined as the time for bleaching from an initial emission rate of 1,000 photons/s down to 500 photons. Furthermore it has a brightness of 53, which is on average. The brightness is defined as the product of the extinction coefficient and the quantum yield at pH 7.4 under ideal maturation conditions<sup>4</sup>. | The weak dimeric flourophor mVenus, with an atomic mass of approximately 27 kDA and a pkA of 6.0, has its absorption maximum at 512 nm and its emission maximum at 528 nm. With a photostabillity of 15, it is one of the fastest decaying flourorphores. The photostabillity is defined as the time for bleaching from an initial emission rate of 1,000 photons/s down to 500 photons. Furthermore it has a brightness of 53, which is on average. The brightness is defined as the product of the extinction coefficient and the quantum yield at pH 7.4 under ideal maturation conditions<sup>4</sup>. | ||
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It is important to keep in mind, that the efficiency of the ssrA-LVA tag is affected by temperature. Therefore it might influence the potential of the desired genetic circuit<sup>6</sup>. | It is important to keep in mind, that the efficiency of the ssrA-LVA tag is affected by temperature. Therefore it might influence the potential of the desired genetic circuit<sup>6</sup>. | ||
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Latest revision as of 16:50, 27 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.
Figure 1: Cyrstal structure of the mVenus reporter protein without the LVA degradation tag. Yellow: β−barrel fold enclosing the fluorophore. Created with the PyMOL Molecular Graphic System. PDB entry, Rekas et al. 2002
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.
Characteristics
mVenus
The weak dimeric flourophor mVenus, with an atomic mass of approximately 27 kDA and a pkA of 6.0, has its absorption maximum at 512 nm and its emission maximum at 528 nm. With a photostabillity of 15, it is one of the fastest decaying flourorphores. The photostabillity is defined as the time for bleaching from an initial emission rate of 1,000 photons/s down to 500 photons. Furthermore it has a brightness of 53, which is on average. The brightness is defined as the product of the extinction coefficient and the quantum yield at pH 7.4 under ideal maturation conditions4.
LVA degradation tag
It is important to keep in mind, that the efficiency of the ssrA-LVA tag is affected by temperature. Therefore it might influence the potential of the desired genetic circuit6.
Sequence and Features
Assembly Compatibility:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
[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] Nathan C Shaner1, Paul A Steinbach & Roger Y Tsien (2005) A guide to choosing fluorescent proteins - Nature Methods
[5] Pabak Sarkar, Srinagesh V. Koushik et al. (2009) Photophysical Properties of Cerulean and Venus Fluorescent Proteins - J Biomed Opt. 2009 ; 14(3): 034047. doi:10.1117/1.3156842Methods
[6] Purcell, Oliver et al. “Temperature Dependence of ssrA-tag Mediated Protein Degradation.” Journal of Biological Engineering 6.1 (2012): 10. Web