Amplicap maxt7 high yield message maker kit

Complementary RNA (cRNA) from ACR1, ACR1 2.0, ACR2, ACR2 2.0, ZipACR, and ZipACR 2.0 were synthesized by in vitro transcription using the AmpliCap-MaxT7 High Yield Message Maker Kit (Epicentre Biotechnologies, Madison, WI, USA), solubilized in nuclease-free water and stored at −20 °C.
Total of 20 ng cRNA of these ACRs were injected into Xenopus laevis oocytes, and the injected oocytes were incubated in ND96 solution (96 mM NaCl, 2 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂, 10 mM HEPES, pH 7.4) at 16 °C for 2–3 days. The photocurrents were recorded with a two-electrode voltage-clamp amplifier (TURBO TEC-03X, NPI electronic GmbH, Tamm, Germany) at room temperature (~25 °C) in standard Ringer’s solution (110 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 10 mM HEPES, pH 7.4). Electrode capillaries (Φ = 1.5 99 mm, Wall thickness 0.178 mm, Hilgenberg, Malsfeld, Germany) with tip resistance of 0.4 –1 MΩ were filled with 3 M KCl for the impalement of oocytes. A USB-6221 DAQ interface (National Instruments, Austin, TX, USA) and WinWCP V5.3.4 software (University of Strathclyde, Glasgow, UK) were used for data acquisition.
The 532 nm and 473 nm laser (Changchun New Industries Optoelectronics Tech, Changchun, China) were used for illumination with light intensities of ~100 µW/mm². The light intensities were measured with a Plus 2 power & energy meter (Laserpoint, Milan, Italy).

Based on the pGEMHE-Cr2c-Cyclop1 construct from our previous experiments [8 (link)], three chimeras were generated by exchanging DNA fragments between Cop5 and Cr2c-Cyclop1 with certain restriction sites. The vector pGEMHE-Cr2c-Cyclop1 was digested with 5′-BamHI and 3′-NcoI. For chimeras 1 and 2, the Cop5 part were amplified with 5′-BamHI and 3′-PpuMI restriction sites by PCR, and the amplified fragments were digested by the two enzymes correspondingly. Meanwhile, the other missing DNA fragment from Cr2c-Cyclop1 part was PCR-amplified and digested by 5′-PpuMI and 3′-NcoI. Finally, two fragments from Cop5 and Cr2c-Cyclop1 were inserted to the BamHI/NcoI cut vector in one reaction. To clone chimera 3, the DNA fragment was amplified from Cop5, digested with BamHI/NcoI and directly ligated to the same vector.
All cloned chimeras were confirmed by complete DNA sequencing. To generate linearized DNA, plasmids were digested by NheI. cRNA was then generated after in vitro transcription by the AmpliCap-MaxT7 High Yield Message Maker Kit (Epicentre Biotechnologies).

cRNA was generated with the AmpliCap-MaxT7 High Yield Message Maker Kit (Epicentre Biotechnologies) using a NheI-linearized pGEM-HE XXM YFP plasmid. Oocytes were injected with 20 ng cRNA and incubated in ND96 solution (96 mM NaCl, 2 mM KCl, 1 mM MgCl₂, 1 mM CaCl₂, 10 mM HEPES and 50 µg/ml Gentamycin, pH 7.4) containing 1 μM all-trans-retinal (short retinal; Sigma-Aldrich), unless indicated otherwise.

The DNA of bPAC-eYFP and Venus-bPAC were cloned into the oocyte expression vector pGEM-HE by USER cloning. All bPAC point mutations were introduced by quick change PCR. The rest tested PAC variants were inserted into pGEMHE within N-terminal BamHI and C-terminal HindIII restriction sites. All the constructs were confirmed by sequencing. After plasmid linearization by NheI digestion, cRNAs were generated with the AmpliCap-MaxT7 High Yield Message Maker Kit (Epicentre Biotechnologies). The following plasmids for cRNA production are available from Addgene: pGEM-HE-Venus-bPAC(F198Y) Addgene #165487, pGEM-HE-bPAC(R278A)-Myc-eYFP Addgene #165488, pGEM-HE-2xLyn-ERex-Venus-bPAC(F198Y)/PACmn Addgene #165489, and pGEM-HE-Glyco-Venus-bPAC(S27A) Addgene #165490. Other plasmids are available by request from S. Gao.

The first 425 aa of CaRhGC which covers the opsin and a part of the predicted coiled-coil sequence, was amplified by PCR with primer pair CaRhGC Kp5F (5′-cGGggtaccgataaggataacaatctccgtgga-3′) and CaRhGC 425Xh3R (5′-ccgCTCgaggatggcatcacagtt-3′), (#1, Supplementary Table 4). The KpnI and XhoI digested inserted to the pGEM-BiFC vector to generate a construct containing: 5′-YC (C-terminal 86 aa of YFP)-CaRh425aa-YN (N-terminal 155 aa of YFP)- 3′. After linearization (NheI), coding RNA (cRNA) was synthesized (AmpliCap-MaxT7 High Yield Message Maker Kit (Epicentre Biotechnologies)). 20 ng cRNA was injected in Xenopus oocytes, incubated in ND96 medium (96 mM NaCl, 2 mM KCl, 1 mM MgCl₂, 1 mM CaCl₂, 10 mM HEPES and 50 µg/ml gentamycin, pH 7.4) containing 1 μM all-trans retinal. Fluorescence pictures were taken 3 days after injection with confocal microscope (Leica DM6000).

The bovine olfactory organ CNG (Bos taurus CNGA2) channel mutant T537S was already used in a previous study with PACα and PACβ from E. gracilis (Schröder-Lang et al., 2007 (link)). The bPAC sequence is as previously published (Stierl et al., 2011 (link)). The SthK channel DNA sequence was synthesized by GeneArt Strings DNA Fragments (Life technologies, Thermo Fisher Scientific) according to the published amino acid sequence (Brams et al., 2014 (link); Kesters et al., 2015 (link)) with codon usage optimized for Mus musculus. The DNA fragments were ligated and inserted into the oocyte expression vector pGEM-HE within N-terminal BamHI and C-terminal HindIII restriction sites. For the fly transgenic vector, the DNA insert was ligated into the KpnI and BamHI restriction sites of the expression vector pJFRC7, instead of ChR2-XXL (Dawydow et al., 2014 (link)).
Sequences were confirmed by complete DNA sequencing (GATC Biotech). Exact DNA sequences of all different constructs are shown in the Supplementary Data Sheet 1. Plasmids were linearized by NheI digestion. cRNAs were generated by in vitro transcription with the AmpliCap-MaxT7 High Yield Message Maker Kit (Epicentre Biotechnologies), using the linearized plasmid DNA as template.

The DNA fragments of FcR1 and FcR2 were amplified by PCR with primers carrying the corresponding restriction sites and digested before being inserted into the oocyte expression vectors pGEMHE (Supplementary Fig. 2) carrying different tags. The sequences were confirmed by sequencing. NheI-linearized plasmid DNA was used for the in vitro generation of complementary RNA (cRNA) following the protocol of the AmpliCap-MaxT7 High Yield Message Maker kit (Epicentre Biotechnologies) for oocyte injection.