...the image can be acquired more quickly. 2-Photon Microscopy 2-Photon Microscopy attempts to solve two ...Fluorescence microscopy. Nature methods 2, 910-919 (2005). PubMed PMID: 16299476. 2. Conchello JA, Lichtman JW. Optical...enhance spatial contrast (i.e. optical sectioning (2) or super-resolution (3). Fluorescence microscopy...photobleaching and a reduction in signal intensity. 2-Photon microscopes restrict excitation (and bleaching...can be seen exiting through the tissue. Therefore, 2-photon allows for deeper imaging into thick tissues...imaging objective that collects fluorescence. Like 2-photon microscopy, only a single focal plane of the...relying on raster scanning as is done in confocal or 2-photon microscopes. Total Internal Reflection Microscopy...

...repository of tools for SAR-CoV-2 research. Two new antibodies, Anti-SARS-CoV-2 Nucleocapsid Protein [mBG17...Anti-SARS-CoV-2 Nucleocapsid Protein [mBG86], target distinct epitopes on the SARS-CoV-2 nucelocapsid ...Anti-SARS-CoV-2 Antibodies here! Terry, J.S., et al. (2021). Development of a SARS-CoV-2 nucleocapsid ...prime editor PE7 Antibodies to facilitate SARS-CoV-2 research New viral vector preps of Voltron2 voltage... incorporates La’s N-terminal domain, PE7 (Figure 2).  PE7 shows enhanced editing efficiencies in a number...editor. Find PE7 plasmids here!     Figure 2: PE7 improves prime editing. A) Schematic of PE7 prime...PMID: 38570691. Antibodies to facilitate SARS-CoV-2 research   By Ashley Waldron Ready-to-use recombinant...

...Cas9 plasmid Bullock and Port pCFD2-dU6:2gRNA dU6:2 BbsI Injection or in vitro transcription Virmilion...Protocols gRNA Design Tools CRISPR Blog Posts The following CRISPR plasmids have been designed for use in ... to a genomic DNA locus and encodes a primer binding site and the desired edits on an RT template. Plasmid...CRISPR plasmids that have been designed for use in Drosophila and other insects. CRISPR... can increase transcription of a specific gene. Design your gRNA sequence to direct the dCas9-activator...

...severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus may acquire mutations to escape ... SARS-CoV-2 infects host cells through binding the human angiotensin converting enzyme 2 (ACE2) cell-surface...target of most anti-SARS-CoV-2 antibodies. In the library, each plasmid encodes the mutated spike protein...protein and a barcode, and is designed so that the SARS-CoV-2 spike RBD is displayed on the yeast cell’s surface...Pooled library to study antibody escape in SARS-CoV-2 Recombinant antibodies for aminoacyl tRNA synthetases...viral preps Mapping antibody escape in SARS-CoV-2 by mutational scanning By Seth Kasowitz As multiple...viral spike protein.   Figure 1. (A) SARS-CoV-2 RBD DMS Library generation and sequencing. From Starr...

...applications. LuxSit-i : An artificial luciferase desined de novo using deep-learning approaches. Split-Luciferase...fusing NanoLuc® and Venus fluorophore to Troponin C Ca 2+ binding domain. Nano-lanterns , Enhanced Nano-lanterns...transfection Feng Zhang 21471 pLenti PGK V5-LUC Neo (w623-2) Firefly PGK Lentiviral expression of firefly luciferase...to that of the fluorophore. Conformationally destabilized Firefly luciferase mutants : A set of three ...three sensors that are increasingly structurally destabilized versions of Firefly luciferase which serves ...dimerization , endosomal escape of cell-penetrating peptides , and more. nLuc/cLuc for split Firefly Luciferase...mammalian systems. ID Plasmid Luciferase Type(s) Description PI 16539 pBV-Luc Firefly Insertion of 5' promoter...

... State University. Since its appearance, SARS-CoV-2 has spread to almost every part of the world manifesting...Rapid detection of 2019 novel coronavirus SARS-CoV-2 using a CRISPR based DETECTR assay Sensitivity:10-...primers that target the E and the N2 gene of SARS-CoV-2. If the Cas12a-sgRNA complex binds to the target sequences... method has been optimized to detect the SARS-CoV-2 virus directly using saliva without having to perform...and visual detection of novel coronavirus SARS-CoV-2 Sensitivity: 4.6-11 copies/microliter of RNA targets...single-molecule sensitive. In the case of HIV-1 and SARS-CoV-2 detection, AIOD-CRISPR without preamplification was...easy-to-deploy, protocol for Cas13-based detection of SARS-CoV-2 genetic material (CREST) Sensitivity: 10 copies of...

...H134R)-EYFP CaMKII ChR2/H134R EYFP Constitutive 1, 2, 5, 9 Deisseroth 26973 pAAV-hSyn-hChR2(H134R)-EYFP....WPRE.SV40 CAG ChR2/H134R mCherry Constitutive 1, 2, 5, 9 Deisseroth 127090 pAAV-CAG-DIO-ChR2(H134R)-eYFP...ChrimsonR (soma-targeted) mRuby2 Cre dependent 9 Adesnik 124603 pAAV-EF1a-DIO-ChrimsonR-mRuby2-KV2.1-WPRE-SV40... ChroME (soma-targeted) mRuby3 Cre dependent 9 Adesnik 124651 pAAV-CamKIIa-ChrimsonR-mScarlet-KV2.1 CaMKII...eGtACR1 (soma-targeted) mRuby3 Cre dependent 9 Adesnik Inhibitory: iC++ 137155 pAAV-nEF-Con/Fon-iC++-EYFP...

... discovery, 2, 952326. https://doi.org/10.3389/fddsv.2022.952326Joubert, M. K., Deshpande, M., Yang, J...various components of the immune system. Figure 2: In-vitro models for immunogenicity prediction of ...drug reaction. This system would somewhat act like a 2-step verification. All approaches have their own limitations...Terry, F. E., Roberts, B. J., Rosenberg, A. S., & De Groot, A. S. (2022). In silico Immunogenicity Assessment...in vivo animal models. In these cases, they are designed to mimic the human lymphatic system or even the...

...either SARS-CoV-2, Adenovirus type 41 (AdV-41), or Adenovirus-Associated Virus type 2 (AAV2) – or some...either 1) a physiological effect of SARS-CoV-2 toward destabilizing one’s natural immune response to Adenovirus...Adenovirus (and/or to its partner, AAV) or 2) a sociological effect of the COVID-19 lockdowns causing a lack...adenoviral vector infected cells does not require de novo viral gene expression: Implications for gene...… Chiu, C. Y. (2023). Adeno-associated virus type 2 in US children with acute severe hepatitis. Nature...viruses infecting humans (from seven species, designated as letters A–G) exist. The life cycle of AdVs...short-lived. It is worth noting that it is possible to design a vector that achieves persistent transgene expression...

...published an article on BioRxiv describing a comprehensive SARS-CoV-2 virus-human interaction network ...comprises all SARS-CoV-2 viral proteins except for Nsp3 and Nsp16, and includes an additional catalytically...all became acutely aware of the threat of SARS-CoV-2. Like many in the science community, we’ve been motivated...network in which we expressed 26 of the 29 SARS-CoV-2 viral proteins in human cells and used these viral “bait...identifying these potential human helpers of SARS-CoV-2 (especially the ones that are druggable) is a key ...just at the beginning of understanding how SARS-CoV-2 interacts with human proteins. For example, we used... joining in this worldwide fight against SARS-CoV-2. As we’ve all been affected in various ways by the...

... non-physiological, and difficult to ignore [1, 2]. 2. Fusion to an obligate dimer or oligomer Several...Biol, 2003. 163(2): p. 257-69. PubMed PMID: 14581454. PubMed Central PMCID: PMC2173526. 2. Zacharias, D....changes that the biosensor is designed to detect resulting in false positives [2, 4]. Solutions to the polymer...an A206K mutation [2]. Addgene has many of these constructs and they are designated mGFP, mVenus, etc....analytical ultracentrifugation [2]. Some affinity assays used in the papers describing the FPs above including... nature are prone to dimerization (i.e. EGFP) [1, 2] or even form obligate tetramers (i.e. DsRed) [3]....form dimers in a cell. The Kd of EGFP is 0.11 mM [2]. Following the simple logic outlined above, you might...

...identification.   Fig. 2: Gel image of Plasmid 61564: pBAMD1-2 (~4.7 kb) digested with SpeI and... at ~3.6 kb and ~1.1 kb (lane 3). Uncut DNA (lane 2) has a band at <3 kb, which corresponds to the supercoiled...formation of plasmid multimers. Cell. 1976 Dec;9(4 PT 2):707-16. doi: 10.1016/0092-8674(76)90134-3. PMID: ...Strausbaugh LD, Gray P, Vartak NB, Brown M, Talbot D, Berg DE. pBR322-derived multicopy plasmids harboring large...dimers in Escherichia coli K-12. Plasmid. 1989 Mar;21(2):138-41. doi: 10.1016/0147-619x(89)90057-7. PMID: ...Plasmids and Chromosomes. Microbiol Spectr. 2014 Oct;2(5). doi: 10.1128/microbiolspec.PLAS-0025-2014. PMID...Behavior of Plasmid DNA. Microbiol Spectr. 2015 Apr;3(2):10.1128/microbiolspec.PLAS-0036-2014. doi: 10.1128...

...-CoV-2 receptor-binding domain. BioRxiv.  https://doi.org/10.1101/2020.04.16.045419.  Wrapp D, De Vlieger...select for optimal sybody candidates. Figure 2: Outline of the process for selection of sybodies ...identify and isolate sybodies targeting the SARS-CoV-2 Receptor Binding Domain found on the membrane-bound...Generation Toolbox kit. Find the Seeger lab's SARS-CoV-2 sybodies! References Geertsma ER (2013) FX Cloning...Zimmermann et al., 2020). This selection platform was designed so that any standard lab can quickly select for...strongly to your target protein. The Seeger lab designed 3 different mRNA sybody libraries for use in the... randomized with a defined set of different trinucleotides (corresponding to different amino acids) in...

...status of MEK1/2 was tracked at 5 minute intervals via Western blot. Phosphorylation of MEK1/2 peaks at just... phosphorylated MEK1/2 levels, suggesting PP1/PP2A is responsible for this MEK1/2 negative feedback. ...PubMed PMID: 28232577. Figures 2-4 were modified from this reference. 2. Regot, Sergi, Jacob J. Hughey...precisely control the timing of kinase activity. 2. Studying negative feedback loops of signaling pathways... studied the negative feedback on endogenous MEK1/2 phosphorylation by creating a short pulse of psRaf1...activity. They did this by exposing psRaf1 to a short 2 minute pulse of 500 nm light (turn on psRaf1) followed...Solution: In Zhou et al, the Lin lab present a generic design for creating photoswitchable kinases and use MEK1...

...Currently available from the Kim lab are 2 presynaptic and 2 postsynaptic targeting mGRASP plasmids. Additionally...bacterial mutagenesis or gene targeting Victor de Lorenzo's lab has engineered a modular mini-Tn5 vector... 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...on channelrhodopsins – Chronos & Chrimson Through de novo sequencing of 127 algal transcriptomes, as well... of three sets of cloning vectors (Level 0, 1, or 2) which can be utilized in three successive assembly...

...create the 2nd-generation lentiviral system (Figure 2). The HIV genes that do remain are very important ...1996 Apr 12;272(5259):263-7. PubMed PMID: 8602510. 2. A third-generation lentivirus vector with a conditional...CH, Humeau LM, Rebello T. J Gene Med. 2013 Feb;15(2):78-82. PubMed PMID: 23322669. 6. Genetic treatment...to sickle cell disease. Hoban MD, Orkin SH, Bauer DE. Blood. 2016 Feb 18;127(7):839-48. PubMed PMID: 26758916... Banasik MB, McCray PB Jr. Gene Ther. 2010 Feb;17(2):150-7. PubMed PMID: 19847206. Additional Resources... but without sustained transgene expression. To design a viral vector system that could do both, Addgene...infect. Finally, the transfer plasmid encoded the desired transgene flanked by HIV-1 long terminal repeats...

...clear and detailed methods and image descriptions, a minimum n = 2, and well labeled high- and low-magnification...indicated they wanted to see more negative data (Figure 2.) At the same time, when scientists who support open...is not only housed, but encouraged.    Fig. 2: Results from a survey conducted by Addgene in 2021...392–400. https://doi.org/10.1038/s41434-018-0028-5 de Backer, M. W. A., Brans, M. A. D., Luijendijk, M....gene therapy trials. Human Gene Therapy Methods, 26(2), 71–76. https://doi.org/10.1089/hgtb.2015.040 Initiative...of negative data.  Negative AAV data is highly desirable  In a survey we conducted to improve our Addgene...every researcher experiences and that it often provides incredibly valuable insight into one’s work. As...

...then packaged into AAV’s protein shell, or capsid; 2) the pHelper plasmid which expresses adenovirus genes...takes ~3 hours to complete: 1 hours hands-on time and 2 hours for the qPCR run and data analysis. Refer to... Special thanks for Dr. Dan Stone and Harshana de Silva Feelixge at Fred Hutchinson Cancer Center for...quantification of adeno-associated virus serotype 2-derived inverted terminal repeat sequences. Human ...therapy methods, 23 1, 18-28. PubMed PMID: 22428977. 2. Choi, V.W., Asokan, A., Haberman, R.A., & Samulski...plasmids to produce AAV: 1) the RepCap plasmid which provides the AAV replication (rep) and capsid (cap) genes...replication; and 3) the transfer plasmid which encodes a transgene of interest that’s packaged into the...

...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...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...Are Inactivated by Human Serum. Molecular Therapy 2:218–222 . https://doi.org/10.1006/mthe.2000.0116 Gutierrez-Guerrero...

...transient mammalian expression 101126 pACP-tag (m)- 2 Vector ACP/MCP-tag Empty backbone for stable and transient...label of their choice according to the methods described by George et al. 2004. The ACP and SFP Synthases...respectively) may be expressed and purified as described previously in Stachelhaus et al. 1998 References...