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...techniques have become indispensable tools for molecular and cell biologists over the last two decades,...light. Long-term light exposure can also lead to cellular phototoxicity, and experimental success can be...fused fluorescent protein. This technique is very similar to the widely used FRET, but it eliminates the ...phototoxicity, they successfully imaged multiple subcellular compartments over several minutes using Nano-...concentrations of the compound can be added to the media. Cellular esterase then converts diacetyl coelenterazine-h...important to verify that this substrate does not alter cellular physiology in your system. Optical sectioning...

...proteins they identified have little sequence similarity to previously identified Cas13a-c orthologs, ...mammalian system. Since Cas13d is functionally similar to previously discovered Cas13 enzymes - what makes...to package in low-capacity vectors like AAV, a popular vector due to its low immunogenicity. But these...their cleavage pattern: EsCas13d produced very similar cleavage products even when guides were tiled across...from the genome, and showed success in models of muscular dystrophy. In this case, Konermann et al. targeted...diversity of CRISPR systems, only time will tell how large the genome and transcriptome engineering toolbox...

...therefore determines the timing of gene expression and largely defines the amount of recombinant protein that ...vectors. Negative inducible promoter pBad is another popular prokaryotic promoter often used for bacterial protein...induced promoters; they show near-zero expression at regular temperatures but can be induced by heat or cold... transcription to take place. Addgene also has popular light-regulated two component systems designed ...between cell types or organisms, promoters are similarly variable. Bacterial promoters only work in prokaryotic...related species from which they were derived. Similarly, the various eukaryotic cell types (mammalian,...

...for plasmid cloning Find our molecular biology reference here Find molecular biology protocols here ... or eukaryotic cells is a common technique in molecular biology and scientific research. There are multiple...material is incorporated into the host’s genome at a particular integration site. The integrated phage remains...material can pass through. If you are starting any molecular biology experiment check out Addgene’s protocol... protocols and videos for techniques in basic molecular biology, plasmid cloning, and viruses. References...

...Addgene.org Addgene's most popular lentiviral plasmids Addgene's most popular retroviral plasmids Addgene's...organisms. This is often cDNA, shRNA, or something similar, and is contained on the transfer plasmid. If expression...enveloped which is achieved by expression of the vesicular somatitis virus G glycoprotein (VSVG). VSVG is...different from gamma-retrovirus, but they perform very similar functions to infiltrate the host cell and deposit.... Viral packaging and entry Adenovirus is quite large and has more helper plasmid components than the ...

...have been derived from. Lentiviruses have slightly larger packaging capacities than gamma-retrovirus, coming...closely related, their required components are very similar. While plasmids containing virus-specific elements...currently known to cause any diseases. Recombinant AAV largely does not integrate, and the addition of a helper...to specific cell types of interest. This is a particularly nice feature as organ-specific injections, or... that of lenti and gamma-retro viruses. This is large enough for many applications, but in some cases ...transient expression of a genetic cargo or have a very large cargo. Fast fact: All viruses discussed in this...

...ends, some 50% of these groups continue to meet regularly, providing ongoing support, advice and accountability...and mentees. Because the participants are at a similar development stage, they have a lot to learn from...Sometimes it is helpful to find peers that share a similar direction or problem. For example, a group of people...most hits. Commit to a meeting schedule and meet regularly no matter what, even if one person can’t make ... work at the same place, a 1-2 hour lunch on a regular schedule might work. If not, perhaps a 3 hour breakfast... handy list of suggestions.  Get started. Have regular meetings. Experiment with the process. Have each...

...can be particularly useful in academic labs when creating shared experimental protocols. Clarify: Asking...setting, where student and postdoc turnover happens regularly and it behooves everyone in the lab to be using...Asking for clarification during a meeting can help get everyone on the same page. Others in the meeting might...communication has helped Addgene move from completing 2-3 large projects a year, on average, to completing 8-10 ...understanding you can be incredibly frustrating. Clarifying the words —> decreasing frustration —> increasing...

...time intensive to process a large number of microbiome samples. To enable large-scale, longitudinal studies...Gram-negative cells.  The Wang lab further developed a modular suite of plasmids (pGT) with various replicative... honey bee microbiome. The toolkit consists of modular parts that are assembled with Golden Gate Assembly...other microbiome systems. Find the toolkit for modular assembly of broad host range plasmids Counting ...bee gut microbiome bacteria with a toolkit for modular assembly of broad-host-range plasmids." ACS synthetic...

...potently inhibit type II CRISPR systems including the popular S. pyogenes Cas9 (Rauch et al., 2017, Hynes et ... on a single plasmid (Grefen and Blatt, 2012). Similar to traditional Gateway™ cloning, the GOI first ... (FRET, Heckler et al., 2015) or ratiometric Bimolecular Fluorescence Complementation (rBiFC, Grefen and...offers a way to spatially and temporally control cellular processes in vivo. Many photoactive proteins exist...photo-reversible protein-protein interactions can control subcellular localization processes in vivo. They deposited...the mitochondria, because cPYP binds to BoPD. A similar set of newly identified light- and dark- binder...

...described above can be used for similar applications and have similar advantages, though with some caveats...chain. Created with biorender.com     The modularity of antibodies means that it is relatively easy..., two full antibodies bound together may be too large. Using a complex that is one full antibody bound...Alternatively, Fab fragments can be used as a block in similar scenarios. By applying unconjugated Fab fragments...and have lower binding affinity than some of the larger fragments.  Think of antibody fragments as new ...

...: IL-36 biology in generalized pustular psoriasis Generalized pustular psoriasis is a chronic autoinflammatory...in this educational video. “SLC6A8 belongs to a large family of solute channels that are responsible for...outside of the cell and translate those signals into cellular responses. For example, GPCRs and their downstream...different receptors upon stimulation. “To study cellular transduction events, we will utilize BRET-, FRET...intensity-based signaling biosensors to monitor intracellular signaling,” says Hébert. Previous research has...

...reagents.  Affinity Reagents  Affinity reagents are molecular tools that have the ability to specifically recognize...affinity reagents is their size. Antibodies are large enough that their bulk can be a barrier at times...difficult to source, may not be readily available for a large number of targets, and can require specific conjugates...   Types of Affinity Reagents  (Listed in no particular order.) Fab fragments are created through enzymatic...variables (scFvs) are ~27kD engineered fusion proteins similar to a Fab fragment but created through engineering...eukaryotic cells, allowing for them to be used as intracellular inhibitors.  Aptamers are not proteins at all...

... we will focus on a unique gene that makes the popular GatewayTM method possible: ccdB. But what is ccdB...start with a brief history of the gene and how molecular biologists have harnessed it to evolve cloning...clones.   ...Becomes a powerful cloning tool Molecular biologists first saw the potential of this system...and are considered F-); however, there are a few popular lab strains, such as NEB Stable, JM109, XL1 Blue...experiments. Please check out Addgene’s Choosing a Molecular Cloning Technique page and the Addgene Blog for...Cloning Posts Resources on Addgene.org Choose a Molecular Cloning Technique Find Plasmid Cloning Protocols...

... to interact by stimulation with blue light.  Similarly, light can also be used to control homo-oligomerization...distribution of clusters without accumulation in subcellular compartments such as nuclear speckles. Furthermore...OptoSTIM1), caused two-fold faster changes in intracellular Ca2+ levels than the original version containing...CRY2clust should prove a useful tool to further probe cellular function. The CRY2clust Plasmid can be found here... commonly observed with direct stimulation are largely absent in the soCoChR-expressing system. Taken ...interactions. We’re excited to see how you adapt similar FLINC biosensors to work in your lab. Find the ...

...encode proteins. They play important roles in many cellular and regulatory processes, but much of their functionality...removing the full ncRNA locus (Ho et al. 2012). For large-scale screening, sgRNA libraries are useful for ... same time (Liu et al. 2018). However, removing large lncRNA fragments with multiple gRNAs poses the risk...Zibitt et al. 2021). Mutating splice sites is particularly effective as it can result in the retention ...Mutations of splice sites. (C) Removal of an exon or a large genomic fragment. (D) Knock-in: insertion of a synthetic...used to target lncRNAs with high specificity, particularly in applications requiring smaller delivery systems...

...developed third option: Fragmid. Fragmid is a modular vector assembly process designed for a quick, plug-and-play...vectors in five days. Did you say… easier? Fragmid’s modular assembly structure takes a lot of the design work... And it truly is plug-and-play. Because of its modular cloning approach, it’s easy to switch out reporters... by phiC31   The Fragmid website While the modular cloning approach is key to the Fragmid system, ..., M. A., Green, T. M., & Doench, J. G. (2024). Modular vector assembly enables rapid assessment of emerging....2024.100519 More resources from Addgene Plasmids 101: Modular Cloning Addgene's CRISPR Guide Plasmids 101: Golden...

... very specific biological targets. However, as popular and useful antibodies are, they are in a midst ...as non-human primates and humans. Since we need larger-volume antibodies for thicker tissues and whole... immunoglobin component that can be produced in large “copies.” This homogeneity circumvents the batch-to-batch...reproducibility and cost-effectiveness for broad and large quantity uses. We therefore use both rAb and mAB...same target to see if the results are the same/similar, emphasizing the antibody’s specificity. Strength...from the background noise. If the sample images similarly to the negative control, that informs us that ...

...and TadA7.10, they designed one set of ABE8’s similarly (ABE8.x-d). In order to assess the effect of removing...you need to modify multiple genes to generate a cellular phenotype. Again ABE8.20-m was the winner. It ...editing in ABE8s While base editors are precise molecular tools, they do have undesired off-target editing...have been described to introduce base changes in cellular DNA and RNA dependent or independent of the gRNA..., and a thoughtful selection of guideRNAs. Of particular note, ABE8s reported in this work did not cause...sites that used to be difficult to target. In particular, their ability for more robust editing in human...

...genes in particular tissues, but they are costly and time-consuming to generate, especially for large animals...Adeno-associated virus (AAV) is a popular tool for gene delivery, but it has a drawback: how do you ensure... it to? Knowing that a gene is expressed in a particular cell type is important not only for translational...types.  Botond Roska’s lab at the Institute of Molecular and Clinical Ophthalmology Basel recently took...Besides restricting AAV transgene expression to particular cell types or classes, these synthetic promoters...