Reagents

The following reagents can be requested from Addgene or directly from us.

BLUE LIGHT-INDUCED HOMO-DIMERIZING LOV DOMAINS
Using database and literature searches, we have identified LOV domains that change oligomerization state in response to blue light stimulation. Human-codon optimized versions of these domains are available tagged to the C-terminus of mVenus and in mammalian expression vectors. A modified Table S1 of our paper in EMBO Journal now including estimated, in vitro dissociation constants and additional references can be found here.
mVenus-VfAU1-LOV (226): LOV domain of Vaucheria frigida Aureochrome 1 [Publication] [Sequence and Construct]
mVenus-CrPH-LOV1 (227): LOV1 domain of Chlamydomonas reinhardtii Phototropin [Publication] [Sequence and Construct]
mVenus-AtPH1-LOV2 (229): LOV2 domain of Arabidopsis thaliana Phototropin 1 [Publication] [Sequence and Construct]
mVenus-NcVV-LOV (231): LOV domain of Neurospora crassa Vivid [Publication] [Sequence and Construct]
mVenus-RsLP-LOV (367): LOV domain Rhodobacter sphaeroides ATCC 17025 photoreceptor [Publication] [Sequence and Construct]
mVenus-NcWC1-LOV (368): LOV domain of Neurospora crassa White Collar-1 [Publication] [Sequence and Construct]

RED LIGHT-INDUCED HOMO-DIMERIZING PHYTOCHROME SENSORY DOMAIN
We repurposed the sensory domain from the cyanobacterial phytochrome 1 (CPH1) of Synechocystis PCC6803 as a new tool to induce protein homodimerization with red light. A human-codon optimized version of this domain is available tagged to the C-terminus of mVenus and in a mammalian expression vector.
mVenus-CPH1S-o (502): Sensory domain of Synechocystis Phytochrome 1 [Publication] [Sequence and Construct follow on Addgene soon!]

GREEN LIGHT-INDUCED MONOMERIZING COBALAMIN-BINDING DOMAINS
We repurposed the cobalamin-binding domain of Myxococcus xanthus and Thermus thermophilus CarH transcription factors as a new tool to induce protein monomerization with green light. Human-codon optimized versions of these domains are available tagged to the C-terminus of mVenus and in mammalian expression vectors.
mVenus-MxCBD (655): Sensory domain of M. xanthus CarH [Publication] [Sequence and Construct follow on Addgene soon!]
mVenus-TtCBD (656): Sensory domain of T. thermophilus CarH [Publication] [Sequence and Construct follow on Addgene soon!]

LIGHT-CONTROLLED RECEPTOR TYROSINE KINASES (Opto-RTKs)
We have engineered receptor tyrosine kinases, e.g. FGFR1, EGFR or RET, that are under light control for optogenetic experiments.
Our first-generation Opto-RTKs were activated by blue light:
Opto-mFGFR1 (204): Blue light-activated murine FGFR1 [Publication] [Sequence and Construct]
Opto-hEGFR (308): Blue light-activated human EGFR [Publication] [Sequence and Construct]
Opto-hRET (317): Blue light-activated human RET [Publication] [Sequence and Construct]
Our second-generation Opto-RTKs are activated by red light (and thus called “r”Opto-RTKs):
rOpto-mFGFR1 (333): Red light-activated murine FGFR1 [Publication] [Sequence and Construct]
rOpto-rtrkB (397): Red light-activated rat trkB [Publication] [Sequence and Construct]
Our third-generation Opto-RTKs are INactivated by green light (and thus called Opto”OFF”-RTKs):
OptoOFF-Mx-mFGFR1 (623): Green light-inactivated murine FGFR1 based on the M. xanthus CBD (see above) under the control of the truncated CMV promoter [Publication] [Sequence and Construct follow on Addgene soon!]
OptoOFF-Tt-mFGFR1 (624): Green light-inactivated murine FGFR1 based on the T. thermophilus CBD (see above) under the control of the truncated CMV promoter [Publication] [Sequence and Construct follow on Addgene soon!]
We have many more Opto-RTKs in the freezer; drop us a line if you are particularly interested in a RTK (family) that is currently not listed here.

RECEIVER PLASMIDS FOR CREATING LIGHT-ACTIVATED RECEPTORS AND OTHER PROTEINS
Based on above domains, we have generated vectors that allow for easy creation of light-activated receptors and light-activated proteins in general. In all vectors, a unique restriction site (AgeI/SgrAI) precedes the domain that is followed by a HA-tag. In addition, vectors contain an N-terminal myristoylation (MYR) membrane anchor or the N-terminal and transmembrane domain (ECD) of the low-affinity neurotrophin receptor p75 with or without a mVenus-tag.
VfAU1-LOV (136): VfAU1-LOV domain preceded by a restriction site, e.g. for insertion of full-length receptors or other genes [Sequence and Construct]
MYR-VfAU1-LOV (135): VfAU1-LOV domain preceded by a MYR signal and a restriction site, e.g. for insertion of receptor intracellular domains [Sequence and Construct]
MYR-CPH1S-o (445): Cyanobacterial phytochrome 1 sensory domain preceded by a restriction site, e.g. for insertion of full-length receptors or other genes [Sequence and Construct]
P75ECD-VfAU1-LOV (448): VfAU1-LOV domain preceded by the extracellular and transmembrane domain of p75NTR and a restriction site, e.g. for insertion of receptor intracellular domains [Sequence and Construct]
mVenus-P75ECD-VfAU1-LOV (458): VfAU1-LOV domain preceded by mVenus, the extracellular and transmembrane domain of p75NTR and a restriction site, e.g. for insertion of receptor intracellular domains [Sequence and Construct]

RECEIVER PLASMIDS FOR CREATING CELL SIGNALING REPORTERS
For the quantification of the MAPK/ERK signaling pathway in our experiments, we designed reporter plasmids containing various reporter genes compatible with our Opto-RTKs under the control of the serum response element. For this we engineered a mother construct containing the response element but no reporter gene:
SRE – AscI (559): Plasmid containing a serum response element (SRE) with an AscI restriction site for reporter gene insertion [Sequence and Construct]

NEW EXPRESSION VECTOR FOR CELL SIGNALING EXPERIMENTS
For proteins that express very well, such as our Opto-RTKs, small vector amounts are typically used in transient transfection experiments if the vector has a strong promoter (e.g. the CMV promoter). For instance, when transfecting HEK293 cells with Opto-RTKs in pcDNA3.1 by lipofection (we use homemade reagents inspired by the Weber-lab [1]), we typically use 50- to 100-fold less DNA compared to our experiments with GPCRs or ionotropic glutamate receptors. This poses a problem for the lipofection method, as large quantities of ’empty’ vector have to be mixed into the reaction to allow for particle formation and reduced cytotoxicity. As a consequence, likely few particles contain vectors with receptors resulting in non-homogenous transfection that can potentially make experiments carried out on the population level (e.g. on all cells in a well of a 96-well plate) difficult to interpret. For trouble-shooting and experiments, we started to use a modified expression vector with a truncated CMV promoter that reduces expression levels significantly and that then can be incorporated at a larger DNA fraction during lipofection (the promoter truncation was originally designed and published by the Mitchinson-lab [2]).
pcDNA3.1(-)-CMVtrunc (620): pcDNA3.1(-) with a truncated CMV promoter following Ref. 2 [Sequence and Construct follow on Addgene soon!]
[1] Müller et al., Nucleic Acids Res. 2013 41(12): e124.   [2] Watanabe and Mitchison, Science 2002 295(5557): 1083.

LIGHT-ACTIVATED G-PROTEIN COUPLED RECEPTORS… COMING SOON!

We believe in sharing of published but also unpublished materials without strings attached.
Drop us a line if you think we might have something that is useful for your research.