Difference between revisions of "Part:BBa K1761004"
(Documentation) |
(non-natural into unnatural) |
||
| (One intermediate revision by the same user not shown) | |||
| Line 26: | Line 26: | ||
The presence of mTurquoise2 was tested with a fluorescence assay. This was done with the construct containing both OmpX and mTurquoise2 and with mTurquoise2 alone. | The presence of mTurquoise2 was tested with a fluorescence assay. This was done with the construct containing both OmpX and mTurquoise2 and with mTurquoise2 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 mTurquoise2 inserted, and pEVOL-pAzF (tRNA + tRNA synthetase). Both vectors were transformed into BL21(DE3). The expression was introduced by adding arabinose, IPTG and the | + | 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 mTurquoise2 inserted, and pEVOL-pAzF (tRNA + tRNA synthetase). Both vectors were transformed into BL21(DE3). The expression was introduced by adding arabinose, IPTG and the unnatural amino acid. |
| Line 39: | Line 39: | ||
<!-- --> | <!-- --> | ||
| − | == | + | == References == |
[1] J. Goedhart et al, “Structure-guided evolution of cyan fluorescent proteins towards a quantum yield of 93%.,”Nature Communications, vol. 3, no. 751, pp 1-9, Mar. 2012. | [1] J. Goedhart et al, “Structure-guided evolution of cyan fluorescent proteins towards a quantum yield of 93%.,”Nature Communications, vol. 3, no. 751, pp 1-9, Mar. 2012. | ||
Latest revision as of 09:36, 18 September 2015
mTurquoise2
mTurquoise2 is an enhanced cyan fluorescent protein, it has a faster maturation, high photostability, longer mono-exponential lifetime and the highest quantum yield measured for a monomeric fluorescent protein.
Usage and Biology
Cyan variants of green fluorescent proteins are widely used as donors in Förster Resonance Energy Transfer (FRET) experiments. The popular, but modestly bright, Enhanced Cyan Fluorescent Protein (ECFP) was sequentially improved into brighter variants, such as mTurquoise2, by Goedhart et al in 2012. mTurquoise2 has a faster maturation, high photostability, longer mono-exponential lifetime and the highest quantum yield measured for a monomeric fluorescent protein. These properties make mTurquoise2 the preferable cyan variant of green fluorescent protein for long-term imaging and as donor for Förster Resonance Energy Transfer (FRET). [1] [2]
Gene Design
We inserted mTurquoise2 in the pETDuet-1 vector together with OmpX and a BsoBI-linker. mTurquoise2 is connected to OmpX with a BsoBI-linker. This linker is a 213 bp long flexible GGSGGS linker and by using the restriction enzyme BsoBI, the linker can become 45 bp shorter. The BsoBI-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). mTurquoise2 is 720 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 mTurquoise2 was tested with a fluorescence assay. This was done with the construct containing both OmpX and mTurquoise2 and with mTurquoise2 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 mTurquoise2 inserted, and pEVOL-pAzF (tRNA + tRNA synthetase). Both vectors were transformed into BL21(DE3). The expression was introduced by adding arabinose, IPTG and the unnatural amino acid.
Fluorescence Confirmation
To confirm whether mTurquoise2 is present in the bacteria, a fluorescence assay was performed. Excitation took place at a wavelength of 420 nm with a laser. The emission spectrum was read out. For more information about how to perform a fluorescence assay, see our Protocol Page [http://2015.igem.org/Team:TU_Eindhoven/Project/Protocols].
References
[1] J. Goedhart et al, “Structure-guided evolution of cyan fluorescent proteins towards a quantum yield of 93%.,”Nature Communications, vol. 3, no. 751, pp 1-9, Mar. 2012.
[2] J. Goedhart, L. van Weeren, M. A. Hink, N. O. E. Vischer, K. Jalink, T. W. J. Gadella Jr, “Bright cyan fluorescent protein variants identified by fluorescence lifetime screening.,” Nature Methods, vol. 7, no. 2, pp. 137-139, Jan 2010.
[3] T.H. Evers et al, “Quantitative Understanding of the Energy Transfer between Fluorescent Proteins Connected via Flexible Peptide Linkers.,” Biochemistry, vol. 45, no. 44, pp. 13183-92, Nov. 2006.