Difference between revisions of "Part:BBa K1761003"
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| − | + | == Usage and Biology == | |
| − | === | + | mNeonGreen is a yellow-green fluorescent protein and is derived from a tetrameric fluorescent protein from cephalochordate Branchiostoma lanceolatum. mNeonGreen is the brightest monomeric green of yellow fluorescent protein yet described and is an excellent fluorescence resonance energy transfer (FRET) acceptor for the newest cyan fluorescent proteins. [1] |
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| + | == Gene Design == | ||
| + | mNeonGreen was characterized by Shaner et al [3]. We inserted mNeonGreen in the pETDuet-1 vector together with OmpX and a BamHI-linker [https://parts.igem.org/Part:BBa_K1761001]. This linker is a 213 bp long flexible GGSGGS linker and by using the restriction enzyme BamHI, the linker can become 45 bp shorter. | ||
| + | mNeonGreen was characterized by Shaner et al [2]. The BamHI-linker is inspired by the article "Quantitative Understanding of the Energy Transfer between Fluorescent Proteins Connected via Flexible Peptide Liners" by T.H. Evers et al from 29 August 2006. [3] | ||
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| + | == Sequence == | ||
| + | The sequence of our mTurquoise2 part has been verified by sequencing at StarSeq. It contains the prefix and suffix with the correct restriction sites (EcoRI, XbaI, SpeI and PstI). mNeonGreen is 711 bp long. | ||
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<partinfo>BBa_K1761003 SequenceAndFeatures</partinfo> | <partinfo>BBa_K1761003 SequenceAndFeatures</partinfo> | ||
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| + | == Characterization == | ||
| + | The presence of mNeonGreen was tested with a fluorescence assay. This was done with the construct containing both OmpX and mNeonGreen and with mNeonGreen alone. | ||
| + | For all the experiments we used the following vectors: pETDuet-1 with a construct inserted (OmpX + intracellular protein) or pSB1C3 with a strong promotor and mNeonGreen inserted, and pEVOL-pAzF (tRNA + tRNA synthetase). Both vectors were transformed into BL21(DE3). The expression was introduced by adding arabinose, IPTG and the non-natural amino acid. | ||
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| + | == Fluorescence Confirmation == | ||
| + | To confirm whether mNeonGreen is present in the bacteria, a fluorescence assay was performed. Excitation took place at a wavelength of 480 nm with a laser. Emission was read out at 517 nm. For more information about how to perform a fluorescence assay, see our Protocol Page [http://2015.igem.org/Team:TU_Eindhoven/Project/Protocols]. | ||
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| + | == Sources == | ||
| + | [1] N.C. Shaner et al, “A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum.,” Nature Methods, vol. 10, no. 5, pp. 407-409, Mar. 2013. | ||
Revision as of 21:44, 9 September 2015
mNeonGreen
mNeonGreen is a yellow-green fluorescent protein. For a fluorescene measurement, the mNeonGreen fluorophore can be excitated with a laser with a wavelenght of 480 nm and readed out at 517 nm.
Usage and Biology
mNeonGreen is a yellow-green fluorescent protein and is derived from a tetrameric fluorescent protein from cephalochordate Branchiostoma lanceolatum. mNeonGreen is the brightest monomeric green of yellow fluorescent protein yet described and is an excellent fluorescence resonance energy transfer (FRET) acceptor for the newest cyan fluorescent proteins. [1]
Gene Design
mNeonGreen was characterized by Shaner et al [3]. We inserted mNeonGreen in the pETDuet-1 vector together with OmpX and a BamHI-linker [1]. This linker is a 213 bp long flexible GGSGGS linker and by using the restriction enzyme BamHI, the linker can become 45 bp shorter. mNeonGreen was characterized by Shaner et al [2]. The BamHI-linker is inspired by the article "Quantitative Understanding of the Energy Transfer between Fluorescent Proteins Connected via Flexible Peptide Liners" by T.H. Evers et al from 29 August 2006. [3]
Sequence
The sequence of our mTurquoise2 part has been verified by sequencing at StarSeq. It contains the prefix and suffix with the correct restriction sites (EcoRI, XbaI, SpeI and PstI). mNeonGreen is 711 bp long.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Characterization
The presence of mNeonGreen was tested with a fluorescence assay. This was done with the construct containing both OmpX and mNeonGreen and with mNeonGreen alone. For all the experiments we used the following vectors: pETDuet-1 with a construct inserted (OmpX + intracellular protein) or pSB1C3 with a strong promotor and mNeonGreen inserted, and pEVOL-pAzF (tRNA + tRNA synthetase). Both vectors were transformed into BL21(DE3). The expression was introduced by adding arabinose, IPTG and the non-natural amino acid.
Fluorescence Confirmation
To confirm whether mNeonGreen is present in the bacteria, a fluorescence assay was performed. Excitation took place at a wavelength of 480 nm with a laser. Emission was read out at 517 nm. For more information about how to perform a fluorescence assay, see our Protocol Page [http://2015.igem.org/Team:TU_Eindhoven/Project/Protocols].
Sources
[1] N.C. Shaner et al, “A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum.,” Nature Methods, vol. 10, no. 5, pp. 407-409, Mar. 2013.