mNeonGreen in a tandem fluorescent timer?

[Richard Mort]

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Hi, I'm wondering if anybody has experience with the above flurophore?
In particular I'm interested in its fast maturation time - has anyone
tried to measure this more accurately - they got it down to below 10mins
in the original paper (Shaner et al Nature Methods 2013). Would it be a
good substitute for sfGFP taking advantage of its yellow shifted
emission and pairing it with Cerulean or mCherry?
Best
R

--
Dr Richard Mort
MRC Human Genetics Unit
MRC IGMM
University of Edinburgh
Western General Hospital
Crewe Road
Edinburgh.
EH4 2XU, UK

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The University of Edinburgh is a charitable body, registered in
Scotland, with registration number SC005336.

[George McNamara]

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Hi Richard,

mNeonGreen and FastTimer are each fast, but are going to be O2 (and
temperature) dependent. If you want really fast, UnaG (Kumagai et al
2013, bonus: reported to be brighter than EGFP etc, and 17 kDa) or
phiLOV (Christie et al 2012) or FluBO (Potzkei et al 2012) are O2
independent and appear to be very fast maturation (phiLOV and FluBO
excite from ~370-480 nm, emit from ~490-550 nm, and ~12 kDa so
brightness could be doubled by making a tandem dimer and still be a bit
smaller than EGFP's 27 kDa). I recently submitted an intramural funding
request for making an O2 biosensor consisting of a localized (nice
bright dot with dark background to localize other biosensors) fusion
protein of UnaG-TALE-Medium Timer (MT has ~1.2 hour half time for
becoming blue, then ~3.9 hour half time becoming red -- both of these
times are with sufficient O2), along with a synthetic variable number
tandem repeat (sVNTR) landing site. Result should look like figure 4a of
Robinett et al 1996 ( http://jcb.rupress.org/content/135/6/1685.long - I
believe the nuclear background smog is due to overexpression), except we
would the FingR approach of Gross et al 2013 and Mora et al 2013 to
eliminate the smog.

with respect to other biosensors, I recommend Newman et al 2011 (if your
library has a subscription, also Newman and Zhang 2014's book chapter
and book) and Frommer's web site
http://biosensor.dpb.carnegiescience.edu/biosensors

Christie JM, Hitomi K, Arvai AS, Hartfield KA, Mettlen M, Pratt AJ, Tainer JA,
Getzoff ED. Structural tuning of the fluorescent protein iLOV for improved
photostability. J Biol Chem. 2012 Jun 22;287(26):22295-304. doi:
10.1074/jbc.M111.318881. Epub 2012 May 9. PubMed PMID: 22573334; PubMed Central
PMCID: PMC3381190.

Gross GG, Junge JA, Mora RJ, Kwon HB, Olson CA, Takahashi TT, Liman ER,

Ellis-Davies GC, McGee AW, Sabatini BL, Roberts RW, Arnold DB. Recombinant probes
for visualizing endogenous synaptic proteins in living neurons. Neuron. 2013 Jun
19;78(6):971-85. doi: 10.1016/j.neuron.2013.04.017. PubMed PMID: 23791193; PubMed
Central PMCID: PMC3779638.

Kumagai A, Ando R, Miyatake H, Greimel P, Kobayashi T, Hirabayashi Y,
Shimogori T, Miyawaki A. A bilirubin-inducible fluorescent protein from eel
muscle. Cell. 2013 Jun 20;153(7):1602-11. doi: 10.1016/j.cell.2013.05.038. Epub
2013 Jun 13. PubMed PMID: 23768684.

Mora RJ, Roberts RW, Arnold DB. Recombinant Probes Reveal Dynamic Localization
of CaMKIIα within Somata of Cortical Neurons. J Neurosci. 2013 Sep
4;33(36):14579-90. doi: 10.1523/JNEUROSCI.2108-13.2013. PubMed PMID: 24005308;
PubMed Central PMCID: PMC3761057.

Potzkei J, Kunze M, Drepper T, Gensch T, Jaeger KE, Büchs J. Real-time
determination of intracellular oxygen in bacteria using a genetically encoded
FRET-based biosensor. BMC Biol. 2012 Mar 22;10:28. doi: 10.1186/1741-7007-10-28.
PubMed PMID: 22439625; PubMed Central PMCID: PMC3364895.

Robinett CC, Straight A, Li G, Willhelm C, Sudlow G, Murray A, Belmont AS. In
vivo localization of DNA sequences and visualization of large-scale chromatin
organization using lac operator/repressor recognition. J Cell Biol. 1996
Dec;135(6 Pt 2):1685-700. PubMed PMID: 8991083; PubMed Central PMCID: PMC2133976.
http://jcb.rupress.org/content/135/6/1685.long

**

Newman RH, Zhang J. The design and application of genetically encodable
biosensors based on fluorescent proteins. Methods Mol Biol. 2014;1071:1-16. doi:
10.1007/978-1-62703-622-1_1. PubMed PMID: 24052376.

Newman RH, Fosbrink MD, Zhang J. Genetically encodable fluorescent biosensors
for tracking signaling dynamics in living cells. Chem Rev. 2011 May
11;111(5):3614-66. doi: 10.1021/cr100002u. Epub 2011 Apr 1. Review. PubMed PMID:
21456512; PubMed Central PMCID: PMC3092831.

Frommer 2013 Molecular sensors
http://biosensor.dpb.carnegiescience.edu/biosensors

My overall concept for Tattletales multiplex fluorescent biosensors is
described at http://works.bepress.com/gmcnamara/26/ (not updated in a
few months, so currently lacking UnaG and FingR) and to take Brainbow (
http://en.wikipedia.org/wiki/Brainbow ) to the subcellular level of
fluorescent "dot codes" I've proposed multicolor address T- and Tumor
rainbow cells, aka T-Bow.

enjoy,


George



On 10/10/2013 7:00 AM, Richard Mort wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi, I'm wondering if anybody has experience with the above flurophore?
> In particular I'm interested in its fast maturation time - has anyone
> tried to measure this more accurately - they got it down to below
> 10mins in the original paper (Shaner et al Nature Methods 2013). Would
> it be a good substitute for sfGFP taking advantage of its yellow
> shifted emission and pairing it with Cerulean or mCherry?
> Best
> R
>

--



George McNamara, Ph.D.
Single Cells Analyst
L.J.N. Cooper Lab
University of Texas M.D. Anderson Cancer Center
Houston, TX 77054
http://works.bepress.com/gmcnamara/26/