Reagents

We believe in sharing of published but also unpublished materials without strings attached. Have a look at receptors, genes and reagents that we developed and drop us a line if you think we might have something that is useful for your research (even if it is not listed here).

The following vectors can all be requested from Addgene or directly from us.

OPTOGENETIC VECTOR COLLECTION (OVC)

Many optogenetic tools are based on light-control of protein-protein interactions. This is achieved by fusing target proteins to naturally occurring light-sensitive domains (LSDs, e.g. LOV domains) that change their oligomerization state (e.g. dimerize) upon illumination. To streamline this process, we engineered a plasmid collection comprised of 38 plasmids to rapidly generate custom-made optogenetic tools for the manipulation of protein-protein interactions [Publication].

The vectors are based on a modular cloning cassette termed ABC containing three insertion sites (A, B, and C; Figure 1A).  Restriction sites A and C (AgeI and XmaI, respectively) generate compatible cohesive ends so that the target sequence can be inserted both N- or C-terminally using only one PCR product (Figure 1A, B). We generated plasmids that contain 1 of 11 LSDs or LSD binding partners in site A as well as separately in site C. We included following LSDs (Figure 1C; these include most of those listed separately in the sections below) [Sequences and Constructs as Addgene Kits]:

  • Blue-light induced homodimerization: VfAU1-LOV, CrPH1-LOV, NcVVD-LOV
  • Blue-light induced oligomerization: AtCRY2-PHR
  • Blue-light induced heterodimerization: AsLOV2-EcSSRA (+EcSspb micro or nano), AsLOV2-pep + HsPDZ1b
  • Blue-light induced dissociation: RsLP-LOV
  • Green-light induced dissocation: MxCarH-CBD, TtCarH-CBD
  • Red-light induced homodimerization: ScPH-1
  • Red-light induced heterodimerization: AtPhyB-S (+AtPIF6)
Figure 1: Optogenetic vector collection. (A) ABC and alternative casette designs. (B) Engineering strategy enables insertion of target amplicon with either AgeI or XmaI overhangs in all LSD vectors. (C) LSDs and their binding partners included in the vector collection.


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]


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]

mVenus-TtCBD (656): Sensory domain of T. thermophilus CarH [Publication] [Sequence and Construct]


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]

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]

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 Mitchison-lab [2]).

pcDNA3.1(-)-CMVtrunc (877): pcDNA3.1(-) with a truncated CMV promoter following Ref. 2 [Sequence and Construct]

[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

Sequence-based OptoXR design methodologies: We have engineered a library of >60 chimeric receptors that contain the signaling domains of human orphan and understudied GPCRs functionally linked to the light-sensing domain of rhodopsin. The chimeric genes can be explored to study the signaling function of human GPCRs that promise to be important regulators of physiology and potential drug targets [Publication]. These are the receptors that were included in the study: GPR1, GPR3, GPR4, GPR6, GPR12, GPR15, GPR17, GPR18, GPR19, GPR20, GPR21, GPR22, GPR25, GPR26, GPR27, GPR31, GPR32, GPR33, GPR34, GPR35, GPR37, GPR37L1, GPR39, GPR42, GPR45, GPR50, GPR52, GPR55, GPR61, GPR62, GPR63, GPR65, GPR68, GPR75, GPR78, GPR82, GPR83, GPR84, GPR85, GPR87, GPR88, GPR119, GPR120, GPR132, GPR135, GPR139, GPR141, GPR142, GPR146, GPR148, GPR149, GPR150, GPR151, GPR152, GPR153, GPR160, GPR161, GPR162, GPR171, GPR173, GPR174, GPR176, GPR182 and GPR183. [Sequences and Constructs]

As controls, we engineered light-activated variants of nine prototypical GPCRs using the same methodology [Publication]: Adrenergic receptors beta2-AR & alpha1-AR, dopamine receptors D1R & D2R, muscarinic acetylcholine receptors M1R, M2R & M3R, adenosine receptor A2A and free fatty acid receptor FFR3. [Sequences and Constructs]

Structure-based OptoXR design methodologies: We have for the first time designed OptoXRs using structural information (GPCR-G-protein contacts obtained from X-ray/CryoEM structures) [Publication]. This was also one of few studies in which the coupling profile of a GPCR was rationally reengineered. [Sequences and Constructs] for the light-activated adrenoreceptors and adenosine receptors are described in the supplementary material of the publication and available from us should you not wish to employ gene synthesis.