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...in bacteria, so many that it may be difficult to decide which path to take. A quick and easy way to introduce... the Lambda bacteriophage, is arguably the most widely and productively used  (Thomason et al., 2014)....In recent years, CRISPR-Cas9 technology has also aided in the creation of scarless mutations, both in stand-alone...What are the counter-selectable markers used to identify double crossovers? The most commonly used counter-selection...the genome. Subsequent selection on sucrose will identify double crossovers. This can be an efficient method...unmarked mutants, but it frequently requires considerable optimization to ensure strong counter-selection...Central PMCID: PMC4862005. Jiang, Wenyan, et al. "RNA-guided editing of bacterial genomes using CRISPR-Cas systems...

...others. Another peptide-based strategy uses an enzyme to covalently label the peptide with a fluorophore... Balla. "Live cell imaging of phosphoinositides with expressed inositide binding protein domains." Methods...University of Amsterdam). GFP is the most popular, most widely used genetically encoded fluorescent probe. Several...proteins as described above. The smallest tags are peptides that are engineered to bind specifically and with...homology domains can be used to detect specific phosphoinositides (Varnai and Balla, 2008) (Figure 2). RNA production...the continuous effort of numerous scientists world-wide.   Many thanks to our guest blogger, Joachim Goedhart...PubMed PMID: 10748019.  8. Lotze, Jonathan, et al. "Peptide-tags for site-specific protein labelling in vitro...

...or developmental characteristics that make them ideal for studying in a laboratory environment. We’ve ...manipulation and genetics research. Plasmids are ideal tools for studying genetic modification for their... E. coli biotin ligase, BirA, has been used to identify and study protein-protein interactions in proximity...pathological developments (Vieira et al., 2019) Besides their ability to regenerate, the axolotl has several...several attractive characteristics that make them an ideal model organism; they are relatively simple to maintain...have large, easily modifiable embryos, which is ideal for observing and manipulating the entire developmental...scientific history, highly characterized genome, and widespread utilization, Arabidopsis has made significant...

...clusters under blue light Check out our Optogenetics Guide to learn more about how these modules work, and ...important components of the cytoskeleton. They provide cells with physical structure, separate chromosomes...microtubules, Opto-katanin will surely be useful for a wide variety of experiments. Opto-RhoGEFs manipulate ...by Rho family signaling pathways. Rho guanine-nucleotide exchange factors (GEFs) activate their partner...system (Mahlandt et al., 2023). These Opto-RhoGEFs provide a reversible and non-invasive way to activate a...efficiency in mammalian cells and in vitro, opening up a wide realm of possibilities. The CRY2-SuperPLD module...membrane or lysosomes (the earlier article also provided CIB constructs for targeting Golgi, ER, and endosome...

...tissue is identifying the cells you are studying. Antibodies can help reveal cellular identities by targeting...while also supporting imaging, making it easy to identify dozens or hundreds of particles surrounding each... A) Schematic of workflow. B) Detail of spacer peptides (3HB: 11-nm 3-helix bundle; SAH: 60-nm single ...with MagIC-cryo-EM. Nucleosome-like particles identified by Topaz software (green circles), magnetic bead...its chaperone NPM2, but the technique should be widely applicable to other targets. The modular system..., M., Kamath, N., … & Weissman, J. S. (2024). Brainwide silencing of prion protein by AAV-mediated delivery...Samelson, A. J., … & Weissman, J. S. (2021). Genome-wide programmable transcriptional memory by CRISPR-based...

...proper formation of the disulfide bond between the VL and VH domains. This bond provides stability and solubility...recombinant antibody. They are ~25 kDa single polypeptides that contain the variable light chain (VL) and... two chains are connected by a flexible linker peptide that is usually 15-20 amino acids long and made...lethal to animals.  In the clinic, scFvs’ size also provides advantages over antibodies (Ahmed et al., 2012...oxidizing periplasm of bacteria to allow for proper disulfide bond formation.  expression in strains of bacteria... concentrations in the eye with minimal systemic side effects. scFvs are part of engineered chimeric antigen...determining regions and frameworks contribute to the disulfide bond independent folding of intrinsically stable...

...spectrometry to identify a mass shift proportional to a single phosphate moiety on the peptide of interest...databases of post-translational modifications, identifies a staggering ~250,000 phosphorylation events ...incorporation of phosphoserine (as described below) provides researchers with a valuable tool for understanding...extension and translation of the pSer-incorporated polypeptide. These three components were shown to effectively...mutation. This emphasizes the advantages of bona fide pSer-incorporation into recombinant proteins as ...between different proteins. Beyond these standard considerations, it is important to determine how much phosphorserine...and non-phospho-bands will give the researcher an idea of the purity of pSer-incorporated to non-phosphorylated...

...Addgene blog Important Considerations When Using AAVs Three Key Considerations For Precise Neuronal Targeting...your next AAV experiment, use these questions as a guide for designing your future injection experiments....injection and detection methods. For example, you can provide location, titer dose (Example: 1E13 GC/mL) and ...experiment, read our blog post on important considerations when using AAVs. Experimental results: These...Frontiers in Neuroanatomy and summarized some considerations for precise neuronal targeting using AAV on...to give proper attribution.  We hope that this provides a clear overview of what you need to submit data...tracing Resources on Addgene.org Read our AAV guide Browse all  AAV vectors Find ready-to-use AAV preps...

....” Subscribers to Scientific America would be considered the interested public and it would be safe to...scientific or science communication. A Tweet is still considered scientific communication if the language is aimed...communication.  Formal education is education that is provided through a structured environment such as the classroom...will.  Informal education is education that is provided, typically ad hoc, for learners who can choose... only used in the field and terminology that is widely used but has a very specific use-case (definition...the context of disease testing, it can still be considered jargon in most other uses. Many laypeople would...communication work does not explicitly exclude people outside of the original recipients. Instead, it targets...

...Park et al., 2019). This provided an engineering road map of sorts, giving an idea of the stiffness the fibers...Resources on Addgene.org Optogenetics guide Chemogenetics guide BRAIN initiative plasmid collection ...cumbersome systems during experiments – neither ideal for the intrepid researcher. There existed, therefore...that could continuously monitor multiple systems. Ideally this tool would be wireless, able to monitor outcomes...important as its capabilities are the practical considerations of actually using it - the device has a rechargeable...indeed stable for continuous monitoring. On the gut side of things, the authors optigenetically and chemically...

...make her work understandable to people both inside and outside the scientific community. We’re excited to...making their own fluorescent protein technologies widely available to the research community. To honor their... proteins in which Dronpa domains flanked either side of an enzyme’s active site. In these fusions, ultraviolet...domains could be placed in amino acid loops on either side of an enzyme’s active site and still block enzyme... will be able to use similar designs to create a wide variety of enzymes and proteins with inducible activity...post. Fatima Enam: Biosensors for human milk oligosaccharides Fatima Enam is currently a graduate student...fluorescent protein-based biosensors for Human Milk Oligosaccharides (HMOs). HMOs are thought to play an important...

...meeting with no idea why you are there. Make the most of everyone’s time by considering the level of meeting...meetings spent on details that do not yet need to be decided. Figure out what you need to accomplish to get .... Team Meetings: For standing team meetings, provide agenda sections for each person to add a bullet...meetings tend to fill the shape of their container. Consider whether you actually need a full hour to achieve...the time down to an hour or less and are able to provide more timely feedback on each other’s projects. ...each of your meeting types (see above). We’ve provided some tips above for what works well at Addgene...FInd Tips on Picking a Research Project Read Other Inside Addgene Posts Resources on the Addgene Website...

...not identical, to their defined recognition sequence. Most enzyme manufacturers make High Fidelity versions...from a colleague down the hall, it is always a good idea to take some time to confirm that you are working...gel correspond to the predicted sizes. Watch this video for a quick overview of how to analyze a restriction...accurately visualize the full size of your construct. Consider buffer and temperature compatibility when digesting...avoid this issue. For your gel: Add ethidium bromide (EtBr) to your gel before pouring it. EtBr binds...your sample settles in the gel well and the dyes provide a visual reference point so you can easily assess...Molecular Biology Reference Find More Protocols and Videos      ...

...Red calcium indicators PinkyCaMP and FRCaMPi Micropeptide killswitch for biomolecular condensates AAV-...Figure 1). Biotinylated proteins were isolated, identified by mass spectrometry, and classified by source...Workflow for cell-surface protein labeling and identification. Reproduced from Wu et al. (2025) under CC ... GECIs, so the team could multiplex PinkyCaMP alongside green fluorescent and optogenetic tools. FRCaMPi... imaging applications, including confocal and widefield imaging, two-photon microscopy, and fiber photometry...https://doi.org/10.1371/journal.pbio.3003048   Micropeptide killswitch for biomolecular condensates By Emily..., 2023). Now, they have adapted the 17-residue peptide that caused nucleolar disruption into a general...

...even CRISPR based gene editing. But, how do you decide which tools to use, and what do you need to know...classes of optogenetic tools, and what factors to consider when choosing one.     Figure 1: Example...or inhibit specific neurons without needing to consider secondary signaling pathways. There are many different...signaling pathways. Light exposure removes the block provided by the LOV domain allowing the pathway to be activated...Created with BioRender.com.   Important considerations for using optogenetic tools Once you’ve selected...specific tool. There are many, many options to consider, and the following factors will help you determine...activate or inhibit a cell in succession and maintain fidelity with the stimulus. For example, if you want to...

... same considerations apply as for standard IF. However, there are a few specific considerations to keep...standard immunofluorescence, but with some extra considerations (Im et al., 2019; McLaughlin, 2019).    ...your needs, your sample, and targets, you might decide to do simultaneous IF instead (Figure 1B). To label...a good place to get started.  One important consideration when deciding whether you can do simultaneous...suitable antibody against your target protein, consider fusing a protein tag to the target and using an...epitope-based strategy such as Anti-6xHis. You could also consider strategies using other types of affinity reagents...interpret. Check out (Jambor et al., 2021) for more guidelines for creating clear and informative figures. Pro-tip...

...we recommend that you use a wide gel comb, run the gel on the slower side, and skip lanes between samples...downstream of the promoter found in the backbone. Ideally, the backbone will contain a variety of restriction...plasmid backbone looks like the one found on the left side of the image below. It has a promoter (blue arrow...single enzyme if your insert is flanked on both sides by that enzyme’s restriction sites, but the insert...Methylene Blue Post Visible Light 100 Ethidium Bromide Pre and Post UV Light 0.5 For more information...addition to a DNA ladder standard, it is also a good idea to run an uncut sample of each plasmid to help with...total DNA in a standard ligation reaction. You ideally want a “recipient plasmid : insert ratio” of approximately...

...engineering designs and ideas ranged from a biological breathalyzer to pesticide breakdown, production ...engineering of biology. Participating schools are provided with synthetic biology lab kits and lesson plans...recognized name in the field of STEM education worldwide. The BioBuilder curriculum is now taught in almost...and teachers can investigate together. Teachers decide which BioBuilder lessons to teach, and how — some...know-how to design/build/test their own project ideas using synthetic biology.Launching with a kickoff...newsletters, pairs teams with STEM mentors, and provides a nationally recognized platform in March to exhibit...Synthetic Biology Blog Posts Resources on Addgene.org Guide to SynBio Assembly Standards Find Plasmids for Creating...

...Addgene provides the scientific community? Katz: The most important resource that Addgene provides is an...about the company and what the goals were and so I decided to join. I’ve been working with Addgene since 2014...distribution of the plasmids and their ability to identify and fulfill a need in terms of helping the scientific...is continuing to look at providing other things besides plasmids to the scientific community through Addgene...perspective, what do you think are important considerations for the future of open science? What about ...exciting about the open science movement is that it provides interesting and unique challenges for intellectual...Addgene blog Learn more about about Addgene in our "Inside Addgene" blog series Subscribe to the Addgene blog...

...cloning strategy? A screen will help you more easily identify successful clones so you have to weed through ...relies on antibiotic resistance. But what about identifying clones that contain your desired insert? This...ampicillin plates containing X-gal allows you to identify cells that took up the vector and distinguish ...at a few screening methods. Blue-white screen A widely used method of screening is the blue-white screen...example, choose enzymes that only cut once on either side of the expected insert. If the plasmid contains ...planning to go this route, check out our protocol video on using restriction digests to analyze plasmids...Sanger sequencing determines the precise order of nucleotides within a DNA molecule, in this case a plasmid...