...CRY2PHR-activator fusion floats freely, depicted in the figure below. Once triggered by blue light, the CRY2 and...31X. By using a slit pattern during blue light exposure (470nm), the researchers showed that expression...created a photoactive Cas9 tool, as shown in the figure below. Their most successful design utilized Magnet...on and off. As one might expect from the modular nature of Cas9, Nihongaki et al. showed that it was possible...reach deeper tissues. Additionally, high intensity exposure can cause DNA damage. Far-red light, however, ...to create photoswitchable spCas9, dCas9, and S. aureus Cas9 (saCas9) proteins, showing the versatility... variant of AcrIIA4 (CASANOVA). As shown in the figure below, in the absence of light, Cas9 is bound by...

...a separate fix/perm cytosolic procedure in that case.     Figure 3: For intracellular labeling...antibody through the biotin-streptavidin interaction (Figure 1a). This interaction brings the fluorophore in...plasmids and biotin plasmids at Addgene!     Figure 1: Indirect detection of target markers can be ...(“anti-rabbit”) region of the primary antibody (Figure 1b). As above, the advantage of this system is ...which would label everything but the CD8+ T cells (Figure 2). Even better, we can use the same fluorophore... gating on the negative population.     Figure 2: Dump gating labels all unwanted cell lineages...membrane but inside the cell. Due to their chemical nature, antibodies cannot penetrate the cell membrane;...

...guest bloggers Shravanti Surest from Iowa State University. Shravanti Suresh is a PhD candidate who investigates...This post was contributed by Shravanti Suresh from Iowa State University. Since its appearance, SARS-...19 and some serological tests to determine past exposure. However, the limited availability of reagents... turn to other technologies like CRISPR. Figure 1: An overview of the general schematic of CRISPR...with streptavidin, the lateral flow strip first captures the streptavidin portion of the probe via biotin...probe that is cut will continue to flow and be captured by an antibody further along on the strip. Highlights...Isothermal amplification is done at one constant temperature, eliminating the need for thermocyclers. This...

...primary is conjugated to a fluorophore (Figure 1).   Figure 1: Comparison between how direct immunofluorescence...confocal   Figure 2: High-level overview of an indirect immunofluorescence procedure with key considerations...within a cell,” “Does this disease change the architecture of my cells,” or “How does this mutation impact...non-fluorescent visualization methods. Stay tuned for a future blog post to learn more about these techniques!.... For this strategy to work, it is important to ensure that you are using secondaries with fluorophores...divided into the several steps outlined below and in Figure 2. For a deeper dive into the steps and techniques...involve multiple smaller steps that all help to ensure that antibodies can access their target and that...

...Symposium and Workshop, organized by Future of Research, Academics for the Future of Science, and the MIT Graduate... More information can be found at http://futureofresearch.org/advocating-for-science-boston-2016/. Sign...Science Symposium Boston 2016 Changes since the Future of Research Symposia Ongoing challenges facing...et al, PNAS 2014; Taylor, eLife, 2016). Several Future of Research (FoR) symposia held across the USA ...science to date. Advocacy opportunities for the future No matter your opinion on these changes, it is ...institutional responses to the FLSA ruling. Similarly, from purely a salary perspective, while this is a move in the...For those advocating in this area, keeping the pressure on for further increases will be challenging, ...

... notes on how lab procedures were actually performed, potentially curtailing future confusion (and hours...community-editable fluorescent protein database ReFigure: Save Scientific Figures into Dashboards and Share Your Insights...reproducibility issues in the biological sciences and featured speakers from academia, industry, nonprofits, ...reproducible if its findings can be predictably applied to future work. Importantly, no one should expect that every...Failed attempts to apply irreproducible results to future work cost both time and money with estimates reaching...doing an experiment wrong. However, even simple procedures like pouring a plate or running a gel have intricacies...initiatives to create more reliable reagents for future research. I’ve talked to many researchers who have...

...factors to consider when choosing one.     Figure 1: Example of an experimental optogenetic setup...activity or gene expression. Experimental output is measured and quantified. Created with BioRender.com. ...either excite or inhibit neuronal activity upon exposure to light within a specific range of wavelengths... be used in tandem with the opsin.      Figure 2: Diagram of excitatory (ChR2) activation by light...engineered receptor that change shape with light exposure to activate them. PORTLs use a similar concept...when a signaling pathway is unknown.      Figure 3: Chimeric OptoXR incorporating rhodopsin undergoes...that may go awry in a disease state.      Figure 4: Example of an intracellular signaling pathway...

...Glypican Collection here! Figure 3: General glypican domain structure. Glypicans are proteoglycans ...— no UTR sequence added to the genome (Figure 4). Figure 4: STITCHR consists of a nicking Cas9 ...the FOS gene promoter and the MYC locus (Figure 5). Figure 5: TurboCas protein labeling compared ...synapses and deep-brain fiber photometry.  Figure 1: Characterization of iGluSnFR4 in mice. A) Scheme...choice. After hearing about researchers' favorite features and limitations of existing backbones, we're excited...viral generation and stable cell line creation (Figure 2). Looking for more? Explore our empty and EGFP...Addgene's own Lentiviral backbone plasmids here! Figure 2: Addgene cloned EGFP into pAG Lenti CMV N-HA ...

...10 minutes, but check with your enzyme’s manufacturer to ensure that you’re cutting for the proper duration... the ligation mixture with backbone alone; 2. Transformation containing ligation mixture with insert and...proper orientation. You then add ligase to the mixture to covalently link the backbone and insert and,... phosphatase) are commonly used. Follow the manufacturer’s instructions.   2. Isolate Your Insert and...unfortunately you can’t just throw the digestion mixtures together. You need to isolate your insert and ...charged DNA through the gel. As indicated in the figure on the left, your digested DNA (and undigested ... the Bitesize Bio Blog and their associated manufacturers websites. These stains require you to either...

... active for greater than 50 generations. Figure 1: Mobile-CRISPRi's modular components. Image from...it integrates into the trnS-leu2 locus.   Figure 2: Introducing Mobile-CRISPRi into bacteria. Top...However, the rate of transfer was variable. They measured transfer rates by comparing the number of colonies...several bacteria associated with human disease (S. aureus, Klebsiella pneumoniae, and P. aeruginosa) in addition...isolated from cheese. Knockdown efficiency, as measured by knockdown of a constitutely expressed RFP reporter...median knockdown of ~40-fold across all species measured. Beyond understanding the basic biology of bacteria...pathogens) were grown in presence of trimethoprim, cultures treated with a folA gRNA were more sensitive to...

...MagLOV’s structure changes under magnetic field. Such changes could be harnessed or enhanced for future MagLOV-based...control of biological systems (Wheeler et al., 2016; Duret et al., 2019). But the field has sometimes seen ...1%) and recovered after the magnet was removed (Figure 1). At first, Andrew and Maria were skeptical. ... since the initial report in 2023.)     Figure 1: Fluorescence intensity of EGFP-FlavinTag in ...change of ~75% in response to the magnetic field (Figure 2); they named this variant MagLOV. Andrew was ...and didn’t need any added cofactors.      Figure 2: Fluorescence intensity and ΔF/F of MagLOV, a...magnet is waved under the sample. The imaged area measures approximately 82 mm across. Video courtesy of ...

...which attract fish for them to prey upon (Figure 1). Figure 1: The sea anemone's red color can be used...aeBlue, amilCP, tsPurple, and gfasPurple) (Figure 2). Figure 2: Chromoprotein expression from bacteria...fluoresce at very low levels. Chromoproteins in nature Compared to the history of fluorescent proteins... chromoproteins lost color overnight in liquid culture, though expressing the chromoproteins from the ...chromoprotein plasmid that combines the most desirable features: intense color, fast maturation, and low fitness...     Chromoproteins for allelic exchange Figure 3: pTOX plasmids expressing amilCP and tsPurple...genetic modification integrates into the genome (Figure 3). Here, the chromoprotein is expressed and gives...

...compositions of Cascade. Although seemingly ubiquitous in nature, they are largely underutilized in research settings...mostly a consequence of Cascade's complexity. Figure 1: Schematic of Class 1 CRISPR types. Class 1 includes... systems utilize with type I-F Cascades. Figure 2: Cascade complex from CRISPR type I-E. Created...tools has been challenging. It can be difficult to ensure comparable expression of all components of Cascade...making cuts every so often. Cutting both strands ensures that there isn’t a template strand left behind ...behind that the cell can use to repair. Figure 3: General overview of the Cascade-Cas3 mechanism. Created...components can make Cas9 easier to work with than Cas3. Future targets Cas3 has the potential to be incredibly...

...green cards and citizenship.   Lauren Celano, CEO Propel Careers. Lauren is passionate about working with...This post was contributed by guest bloggers Lauren Celano of Propel Careers and Rachel Casseus, Esq. ...and on. As you think about your career (and your future in general), keep in mind that you may have years...careers - being aware of this can help take the pressure off having to find the perfect job immediately...but not limited to biotechnology, chemistry, architecture, engineering, mathematics, physical sciences...bachelor's degree or its equivalent and state licensure, if required to practice in that field. H-1B work-authorization...background, expertise, publications, and plan for future activities; including the interest of the greater...

...Martin, B., & Shendure, J. (2022). Precise genomic deletions using paired prime editing. Nature Biotechnology...editing world. The approach, originally published in Nature, is a “search-and-replace” genome editing technique... breaks (DSBs) or donor DNA templates.  Figure 1: An overview of prime editing. Created with BioRender.com...Liu lab contributed an improved prime editing “architecture” that can be combined with any of the PE2-PE5...prime editing as efficient as possible. Figure 2: Decision tree for selecting a PE6 variant. PE...2023, under a CC BY 4.0 license. epegRNAs: Structured RNAs with improved stability Traditional sgRNAs...of large DNA sequences with twin prime editing. Nature Biotechnology, 40(5), 731–740. https://doi.org/...

... 1) follows this structure: Step 1: fix (and maybe label) your sample (i.e. cultured cells or tissues...and after expansion and measure the degree of agreement between them to ensure the expanded images are...how you interpret a target’s structure. For example, cytoskeletal structures that form continuous lines ...easier, more accessible way to image biological features previously only visible via techniques like super-resolution...by just adding water.)  Step 5: Image   Figure 1: The ExM process allows you to physically magnify...depend upon the specific gel recipe used, ambient temperatures, incubation times, and the physical properties...properties of the sample itself. For example, cultured cells are typically easier to expand than mouse brain...

...Template). Nature Protocol Exchange Website: http://www.nature.com/protocolexchange/ Nature Protocol Exchange...for iOS and Android). A neat feature of protocols.io is the checklist feature which allows you to check ...process so you’ll have a record of your work. This feature further simplifies lab work if you like to bring...an open online protocol repository hosted by the Nature Publishing Group. Like the previously mentioned...and create lists of your favorite protocols for future reference. The exchange currently boasts over 2,500...links to protocols on their own websites. We're not sure how frequently it's being updated now, but there...access to protcols from external vendors, such as Nature Protcols and NEB. Quartzy Website: https://www....

...survive high temperatures (up to 95 °C) and is active over a range of elevated temperatures (37–75 °C)....elevated temperatures, allowing further discrimination by incubating the ligation at a temperature near the...Reaction Temperature Calculator can be used to estimate an appropriate incubation temperature when using...will also efficiently ligate many undesirable structures, including substrates containing gaps or DNA ...phosphorylated base can be observed in the crystal structures of several DNA ligases bound to substrate (23... the melting temperature (Tm) of the DNA strands. This selectively reduces the concentration of annealed.... 2. Tomkinson, Alan E., et al. "DNA ligases: structure, reaction mechanism, and function." Chemical reviews...

....  After this, lowering the temperature allows the repressor to re-nature and again repress expression...red recombineering system has three components (Figure 1): 1) Exo, 2) Beta, and 3) Gam. All three are ...recombineering with an ssDNA oligo substrate.   Figure 1: Components of the Lambda Red Recombineering ...a generic lambda red recombineering experiment (Figure 2). In the following sections, key steps that differ...confirmation of recombinant clones.    Figure 2: Overview of using Lambda Red recombineering ... pairs of homology to the targeted insert site (Figure 2, top).  These primers are typically ~70 nucleotides...few strains can also be found at Addgene, but be sure to read the associated papers for their particular...

... guest blogger Jack Leeming Editor of Naturejobs, part of Nature magazine. Humans are built to enjoy, ... your structure. Develop your protagonist In most cases, the protagonist will be you. Make sure to highlight...accept and acknowledge your failures. Foster conversation A story is not a lecture. Make yourself open to ... parts of our lives that tend to have a story structure: beginning, end; cause, effect; lesson, learning...apologised and admitted he’d been under a lot of pressure recently which had affected his judgement. Eventually...rhetorical technique you have in your arsenal to make sure people pay attention. Highlight your lowest points... discovery into a scientific story, but it will ensure that people pay attention. Practice Nobody gets...