...fall under two general categories: rho-dependent and rho-independent. Rho factor is a helicase which assists...polymerase in the termination of the transcript. Rho-dependent terminators are not usually employed in...Nearly all common bacterial expression plasmids use Rho-independent terminators, which include naturally ...engineered high-efficiency terminators such as T0. Rho-independent termination is also known as intrinsic...conserved secondary structure and sequence conserved Rho-independent termination annotation for 90 bacterial...structure to induce transcript cleavage, similar to the Rho-independent termination found in prokaryotes. This...Polymerase I uses a process similar to the prokaryotic Rho-dependent mechanism, whereas Polymerase II termination...

....    Figure 2: Opto-RhoGEFs to control Rho GTPase activity. A) Schematic of Opto-RhoGEF design and activity...movement, and more, often driven by Rho family signaling pathways. Rho guanine-nucleotide exchange factors...GEFs ITSN1, TIAM1, or p63RhoGEF (which activate the Rho GTPases Cdc42, Rac1, and RhoA, respectively) using...depending on the specific Rho GTPase/GEF pair (Ridley, 2016). Taking control of a RhoGEF is a great way to stimulate... J. (2023). Opto-RhoGEFs, an optimized optogenetic toolbox to reversibly control Rho GTPase activity on...factors (GEFs) activate their partner Rho GTPase, leading to behaviors like lamellipodia or filopodia formation...membrane-recruitment optogenetic tools for manipulating Rho GTPases based on BcLOV4. These single-component systems...

...target sites for editing. PAM sequences can be from 2-6 base pairs in length and follow the DNA sequence... Eric Perkins Mammalian genomic DNA is not just a 2-D linear molecule snaking through our cells’ nuclei...PubMed PMID: 30166441. MerMAIDs - marine channelrhodopsins for the optogenetic toolbox Article contributed...contributed by Susanna Bachle Anion-conducting channelrhodopsins (ChR) are ion channels that can be used to...

...still depolarize neurons at lower MOIs. Figure 2: ChRgers are delivered by intravenous injection into... shines on excitatory channelrhodopsin-expressing neurons, the channelrhodopsins respond by opening and...deliver channelrhodopsins to the brain. This delivery route concentrates lots of channelrhodopsins in one...since current channelrhodopsins tools have low conductance, but also confines channelrhodopsin activation...conductance compared to current channelrhodopsins. Their new channelrhodopsins, called ChRgers, make the brain...engineering channelrhodopsins There are three main challenges to engineering channelrhodopsins to have improved...to predict if a channelrhodopsin sequence would result in a functional channelrhodopsin, as well as predict...

... L. Direct multiplex imaging and optogenetics of Rho GTPases enabled by near-infrared FRET. Nat Chem Biol... iRFP713, % Oligomeric state Photo-stabilityc, t1/2, s pKa Brightness in HeLa cells vs. iRFP713, % d ...blue-light controlled optogenetics, such as channelrhodopsins and tools based on light-oxygen-voltage (LOV...

...GEVIs (called ASAP 1 and 2), they found an indicator that is optimized for 2-photon imaging of voltages...resultant JEDI-2P sensors overcome many challenges in 2-photon in-vivo imaging of voltage fluctuations. Image...suitable for in vivo optogenetic use.   Fig. 2: A) HcKCR1 is highly selective for K+ over other Na...slice of HcKCR1-EYFP and tdTomato expressed layer 2/3 neurons in mouse. C) Action potential frequencies... of light-gated potassium channelrhodopsins, named Kalium channelrhodopsins (KCRs), from a fungus-like...-2P Novel class of light-gated potassium channelrhodopsins Myc-tag antibody anti-c-Myc [9E10] now available...   Novel class of light-gated potassium channelrhodopsins by: Mike Lacy New work from the labs of Mingshan...

...stimulation         In the video above, Channelrhodopsin 2 (Chr2) has been expressed in the motor cortex... example, the light-gated cation channel, Channelrhodopsin 2 (ChR2), exists in several states: closed/...different steps and materials required for each [1, 2]. This protocol [3] provides, in exquisite detail,...lasers are relatively cheap and will run you about $2-3,000/laser. However, an important caveat is that ...PMID: 21628464; PubMed Central PMCID: PMC3138296. 2. Sparta DR, et al. Construction of implantable optical...board, Jacqueline N Crawley  [et al]. 2012;Chapter 2:Unit2 16. doi: 10.1002/0471142301.ns0216s58. PubMed...coordination by Gi/o protein-coupled vertebrate rhodopsin in cerebellar Purkinje cells. The Journal of biological...

...light-activated tool for controlling neuronal activity, channelrhodopsin-2 (ChR2), a blue light-activated cation channel..., D., Hegemann, P., & Bamberg, E. (2003). Channelrhodopsin-2, a directly light-gated cation-selective ...used in tandem with the opsin.      Figure 2: Diagram of excitatory (ChR2) activation by light ...) domains, phytochrome B (PhyB), and cryptochrome 2 (CRY2) (Zhang & Cui, 2015). When stimulated with light...comparative analysis of microbial opsins. Nature Methods, 9(2), 159–172. https://doi.org/10.1038/nmeth.1808 References...comparative analysis of microbial opsins. Nature Methods, 9(2), 159–172. https://doi.org/10.1038/nmeth.1808 Nagel...intracellular signaling pathways. Trends in Biotechnology, 33(2), 92–100. https://doi.org/10.1016/j.tibtech.2014.11.007...

...PMC3337962. 8. Zhao S, et al. Cell type-specific channelrhodopsin-2 transgenic mice for optogenetic dissection...commonly used in optogenetics: 1) electrophysiology and 2) behavioral experiments. I’m not an electrophysiologist...choice in the brain region you wish to stimulate 2) Fabrication and implantation of the optical fiber...thoroughly elsewhere (here and here for instance) [2], [3]. Karl Deisseroth of Stanford University , one...reviews for a discussion of the different variants [1, 2, 4]. And, of course, the best way to learn about the...monitor expression at various time points (e.g. every 1-2 weeks over 8 weeks) in order to determine the optimal...(4):251-66. doi: 10.1038/nrn3171. PMID: 22430017. 2. Fenno L, et al. The development and application of...

...Currently available from the Kim lab are 2 presynaptic and 2 postsynaptic targeting mGRASP plasmids. Additionally... cells were pulsed with 2µg/ml of Doxycycline for 2 hours, and imaged on the Keyence BZ-9000 fluorescence...work! Robinson et al., Dev Cell 2010 Feb 16;18(2):324-31. Cheeseman et al., J Cell Sci 2013 May 1...promotes the formation of an isopeptide bond between 2 peptide tags, SpyTag and KTag. The group demonstrated... of three sets of cloning vectors (Level 0, 1, or 2) which can be utilized in three successive assembly...1 modules can be similarly assembled into a Level 2 vector, thus forming a functional genetic circuit....circuit. Flexibility has been built into the Level 2 vectors to allow for additional iterations of Level 1 assembly...

...promoter upstream of an opsin gene (e.g., channelrhodopsin-2, ChR2) fused to a reporter gene (e.g., mCherry...Atasoy, Deniz, et al. "A FLEX switch targets Channelrhodopsin-2 to multiple cell types for imaging and long-range...the correct orientation to be transcribed (Figure 2) (Sharma and Zhu, 2014). How does FLEx enable strong... Abdallah, Khaled, et al. "Adeno-associated virus 2/9 delivery of Cre recombinase in mouse primary afferents...Microbial rhodopsins are encoded by opsin genes, and include bacteriorhodopsins, halorhodopsins, and channelrhodopsins... 50 years ago with the discovery of microbial rhodopsins, light-sensitive proteins that absorb photons...light (Zhang et al., 2011). What do microbial rhodopsins and neurons have in common? Neuronal stimulations...

...fast access to the tools required to study SARS-CoV-2, but with many academic research labs closed during...biobanks, are crucial for the advancement of SARS-CoV-2 research because they provide rapid access to validated...Many of the reagents used to investigate SARS-CoV-2 were developed for broad use and are now important...susceptibility of cells from different organisms to SARS-CoV-2 (Chu et al., 2020). An additional cell line used in...that science is truly a global endeavor. Case study 2: Plasmids Another example of reagents that have broad...applications but have proven useful to the study of SARS-CoV-2 are the lentiviral production and packaging vectors...that characterized the spike (S) protein of SARS-CoV-2 (Ou et al., 2020). The authors requested these plasmids...

...host range plasmids. FEMS Microbiology Letters, 348(2), 87–96. https://doi.org/10.1111/1574-6968.12241  ...mobilization of broad host-range IncQ plasmids. Plasmid, 62(2), 57–70. https://doi.org/10.1016/j.plasmid.2009.05.001...negative hosts. Biotechnology and Bioengineering, 106(2), 326–332. https://doi.org/10.1002/bit.22695  Schmidhauser...Pseudomonas spp., Rhizobium spp., Rhodopseudomonas spp., Rhodospirillum spp., Shewanella spp., Thiobacillus...leguminosarum, R. meliloti, Rhodobacter sphaeroides, R. capsulatus, Rhodopseudomonas spheroides, Salmonella...Rhizobium meliloti, R. leguminosarum by. viciae, Rhodobacter sphaeroides, Salmonella typhimurium, Vibrio cholerae...

...divided up structurally into 2 main groups: voltage sensitive domains and Rhodopsin containing GEVIs. Chemigenetic...depending on your experimental system.   Figure 2: General overview of chemigenetic tools. A genetically..., the JEDI-2P is an excellent choice. Rhodopsin GEVIs Rhodopsin is composed of an opsin (a GPCR) linked...are two flavors of rhodopsin GEVIs. The first is a two-in-one system where rhodopsin functions as both ...second type of these GEVIs: a FP-fused rhodopsin. FP-fusion to rhodopsin resulted in electrochromic FRET capable..., with rhodopsin quenching the added fluor for this two-part fluorescent system, and rhodopsin still functioning...exceeds that of the VSD GEVIs. However, since rhodopsin proteins were initially evolved for ion transport...

... Opto-Cre-Vvd is fast and can cut in as little as 2 hours.  Sheets, MB, et al., ACS Synth Biol. 2020....they investigated three Casɸ orthologs, Casɸ-1, Casɸ-2, and Casɸ-3. This system is notable for several reasons...a minimal PAM requirement. For instance, for Casɸ-2 the PAM sequence is 5’-TBN-3’ (where B is G, T, or...deposited by Leopold Parts. This library delivers 2 randomly paired guides per construct and allows a ...AAV-flex-taCasp3-TEVp here The red-shifted channelrhodopsin (C1V1) targeting parvalbumin cortical interneurons...use viral prep in serotype AAV5.  Get the channelrhodopsin here Astrocyte specific tight affinity glutamate...

...for retrograde transport has been found for AAV1, -2, -5, -7, -8 (Taymans et al., 2007), AAV1 (Hollis et... tropism and axonal transport of AAV serotypes 1, 2, 5, 6, 8 and 9 in the mouse brain. PLoS One 8:e76310...reveal novel hippocampal CA2 circuits. Nat Neurosci 17(2), 269–279. PubMed PMID: 24336151. PubMed Central PMCID... Mechanisms of Motility and Regulation. Neuron 84(2), 292–309. PubMed PMID: 25374356. PubMed Central PMCID...transduction efficiency of recombinant AAV serotypes 1, 2, 5 and 8 in the rat nigrostriatal system. J Neurochem...analysis of adeno-associated viral vector serotypes 1, 2, 5, 7 and 8 in mouse brain. Hum Gene Ther 18, 195–...retrograde access to projection neurons. Neuron 92(2), 372–382. PubMed PMID: 27720486. PubMed Central PMCID...

...safely. Recently, many SARS-CoV-2 researchers began pseudotyping the SARS-CoV-2 spike protein into a lower ...SARS-CoV-2 virus. Scientists have been using similar pseudotyping methods with other viruses in a BSL-2 lab... et a., 2018).   Figure 2: To pseudotype with the SARS-CoV-2 spike protein, scientists encode...Therapy 2:218–222 . https://doi.org/10.1006/mthe.2000.0116 Gutierrez-Guerrero A, Cosset F-L, Verhoeyen E (...production uses three plasmids: (1) The transfer plasmid, (2) the envelope plasmid, and (3) the packaging plasmid...example, production of vesicular stomatitis virus, HIV-2, and human foamy virus in cell lines expressing galactosyl...for Pseudotyping Lentiviral Particles with SARS-CoV-2 Spike Protein for Neutralization Assays. Viruses 12...

...NGG)  Editing window ~2 nt  85175  pBK-EE-BE3  SpCas9 (NGG)  Editing window ~2 nt  85176  pBK-YE2-...quantum yield of 0.54) and mScarlet-H with M164H (2-fold improvement in photostability compared to mScarlet...SpCas9 base editors with editing windows as small as 1-2 nucleotides! To reduce off-target effects associated...pBK-YE2-BE3  SpCas9 (NGG)  Editing window ~2 nt  85177  pBK-YEE-BE3  SpCas9 (NGG)  Lower on-target activity...activity than BE3; editing window ~1-2 nt  87438  pET42b-HF-BE3  HF-Cas9 (NGG)  Very low off-target activity...Ladders Are Awesome Tools: They are comprised of 2 plasmids, pPSU1 and pPSU2, that can be amplified affordably...libraries The Gross Lab at UCSF recently deposited 2 Bacillus subtilis libraries with Addgene – the B. ...

...expressed as a fusion with your favorite protein and (2) a synthetic fluorophore-contianing “ligand” that ...cell 58.4 (2015): 644-659. PubMed PMID: 26000849. 2. Grimm, Jonathan B., et al. "A general method to improve... classic small molecule fluorophores, such as rhodamines (Figure 2a), were discovered in the 19th century...structure have yielded commercial panels of advanced rhodamine fluorophores, such as the Alexa Fluor and ATTO... brightness and photostability of the classic rhodamines and their analogs without sacrificing their small...advanced imaging modalities. Follow him on twitter @rhodamine110.   References 1. Liu, Zhe, Luke D. Lavis, and...

... Sternson SM. 2008. A FLEX switch targets Channelrhodopsin-2 to multiple cell types for imaging and long-range...conditional knock-in cassette. As shown in Figure 2, the cassette should contain a reporter DNA sequence...a targeted neuronal population.    Figure 2 (modified from Schnutgen et al. Nat Biotech 2003):...cellular level in the mouse. 2003 PubMed PMID: 12665802. 2. Branda, CS and Dymecki, SM. Talking about a Revolution...