...customization with strain-specific promoters, antibiotic resistance selection markers, gRNAs, and dCas9 proteins....range of bacteria. Once transferred, Mobile-CRISPRi stably integrates into the genome and is propagated during...Mobile-CRISPRi does not disrupt gene function and is stable and active for greater than 50 generations. ...40-fold across all species measured. Beyond understanding the basic biology of bacteria, Mobile-CRISPRi...Mobile-CRISPRi can be used to identify antibiotic resistance genes. Genes that are targets of antibiotics are often... guides targeting non-essential genes remained constant, with fitness effects more pronounced at 12 doublings...dCas9. Following integration, Mobile-CRISPRi is stably expressed with no selection required and titratable...

...functional tests. The newest genome engineer sharing the stage with much-lauded CRISPR-Cas9 is DNA-guided endonuclease...NgAgo is faring with users in a minute, but, to start, let's review why NgAgo is in the spotlight and ...difficult” GC-rich regions of the genome, which seem resistant to Cas9 cleavage (1). Both the CRISPR-Cas9 and...sequence might not always be available at an optimal distance from the target site. For example, NGG may not...genome editing applications. So, how does NgAgo stack up thus far? A few weeks ago, I was going through...and fussy to optimize. One reason could be the instability of the supposedly alien 5’-phosphorylated ssDNA...optimizing genome-editing methods in brain cells and understanding brain tumor biology. When not snipping DNA, ...

...heavy chain, light chain, constant region, variable region   The constant region of the heavy chain..., or antigen-binding region, and the Fc, or crystallizable region. Two immunoglobulin (Ig) heavy chains...by changing certain amino acids to improve its stability. Additionally, the choice of vector can impact... your sample, you’ll compare your samples to a standard curve. This curve is generated using a serial ...protein that is also detected in the same way. The standard curve that is created from the OD and concentration...antibodies to detect proteins is referred to as staining. This panel of antibodies is used to both identify...Similar to flow cytometry, you’ll use antibodies to stain for markers of one or more specific cell types, ...

... So what’s a researcher to do if they want to understand the ultrastructural relationships that influence...expansion.  In some cases, labels are added after this stage.  Step 4: Expand! (Typically by just adding water...with biorender.com Steps 2-4 are not found in standard IHC/ICC protocols and you might expect them to...common reagents, straightforward protocols, and standard microscopes sounds great, right? But there is ...post-expansion, but that doesn’t mean we should start re-conceptualizing actin filaments or microtubules...scene, ExM has helped researchers gain a deeper understanding of neuronal synapses, centrosomes, parasites... Royal Soc 380:20210110. https://doi.org/10.1098/rsta.2021.0110  Sherry DM, Stiles MA (2022) Improved ...

...Chemical fluorophores can be substantially brighter and more photostable than fluorescent proteins, providing... azetidine rings could substantially enhance the brightness and photostability of the classic rhodamines...enhance small molecule fluorogens (Figure 1e). Stains – These labels consist of a fluorophores conjugated...classic, net-neutral dye scaffold due to its established chemistry, small size, brightness, and cell permeability...In past decades, further refinements to this established dye structure have yielded commercial panels ...Fluor and ATTO dyes, with improved brightness, photostability, and spectral range. Nevertheless, these fluorophores...

...linearized plasmid standard. However, even when the standard is made properly, it is unstable and may only ...the viral DNA to the threshold fluorescence of a standard - a set of reactions with known quantities of ...a known titer, can be used to confirm that the standard curve is giving an accurate readout of your samples... range. We have also found that generating the standard curve for qPCR can be challenging to get right...Droplet digital PCR (ddPCR) does not require a standard or a reference, meaning saving on reagents (and...to contamination. Optimize your PCR For a good starting place on what PCR parameters to use, see Lock’...

...-person events and venues will have a solid understanding of their audience demographics. Do your research...can vary depending on factors like socioeconomic status and geographic location. For example, not all social...Before you embark on a project, do your best to understand what kind of resources - including your time ...specialized equipment and/or expertise from the start, or if you can begin simply and build up to more...potential of your preferred platform before you start can save a lot of trouble in the long run.  Selecting... your audience. Digital or print distribution.  Static. Can be difficult to convey an entire story or ...omitting details. Digital or print distribution.  Static. Often requires both artistic and scientific training...

...plate. You can use this plate to start liquid cultures. Lastly, you can start small overnight liquid cultures...detect the presence of your insert; 2) set up a standard PCR reaction (primers, dNTPs, polymerase) using...PCR reactions is nearly identical to preparing a standard PCR reaction: combine template, primers, polymerase...polymerase, and dNTPs and then incubate with a standard PCR thermocycling program. One key difference is ...leftover bacteria-water suspension and use them to start cultures of your positive clones. If you want to...the initial heating step of the PCR reaction. A standard Taq polymerase is sufficient. Controls: Controls...Make sure to use an appropriate molecular weight standard for reference and to add a loading dye with glycerol...

...regulations, differing customs processes, and the distances the packages needed to travel meant the accessibility...using couriers that refill dry ice if the package stays in transit for a certain number of days and tracking...Addgene distributors Distributor Partnership Start Date Country Served Beijing Zhongyuan 2012...information about the materials in our repository. We started to increase accessibility to our repository by ...types, and more, all of which help a researcher understand if a material is right for their experiment.   ...introduced guides — practical primers to help users get started with tools like CRISPR, lentivirus, and optogenetics...darker blue indicates more views from that country. Stats and figures are helpful, but our favorite measure...

... for numerous non-academic careers. Still, many start their scientific careers hoping to follow the traditional...Possible roadblocks only become obvious at rather late stages (i.e. postdoc level) and can include the pressure...candidates are highly experienced but often have to start pursuing other options. While some non-academic ...existed, but did not consider them for myself. I started picturing myself in a technical support role only...possibly this same reason, it can be one of the more constant and safe corners in a company, less exposed to...where someone tries to “optimize” a particular standard lab protocol. For example, someone may attempt...then complain about high background. In many instances, variations of the product’s specific application...

...and some rising stars in the world of microbiology, some before they had even established their own labs...Tweet - The conference hashtag I had, however, started a Twitter account, and was following the meeting...Favorable conditions - Expansion of networks The last stage is Exponential Growth, named after the growth phase...variability was organic, as some people need less assistance than others, but we did have some participants...worth addressing. Clear expectations have been established for future participants regarding the activities...mentoring. For example, Goldschmidt conference, Coastal & Estuarine Research Federation (CERF) Biennial... and microbiology for the last two decades as a staff scientist, but before going to graduate school, ...

...unintentionally remove connecting pathways between other distant regions. They also don’t give any clues about the...Anxiety tests Rodents have a natural preference to stay in dark areas (protected) as opposed to brightly...mouse can move freely on a 4-arm cross and choose to stay on an open arm (risky), or a closed arm (safe). ...On the other hand, the control group preferred staying in the dark compartment (Howerton at al. 2014)....floating on the water surface. The latter reflects a state of behavioral despair, and the more time the animal...genetic manipulations with the forced swim test understanding the genetic background of depression.  One such...memory. We’ll cover these in a  future blog post, so stay tuned!  References Alpár, Alán, et al. "Hypothalamic...

...human genes have a counterpart in zebrafish. Understanding the function of these genes in fish will help...functions for genes, and lays the groundwork for understanding the gene networks that regulate processes from...mutagenesis screens provided a major toehold into understanding how genes control biology (a fact celebrated...for generating transgenic zebrafish are well established (Felker and Mosimann, 2016). One common and robust...zygote transforms into an organism with a clearly established body plan including head, eyes, and tail. Compared...different methods have been developed, relying on established reagents such as CRISPR/Cas9, Tol2, and the high-quality...profiled 38,731 cells from 694 embryos over multiple stages and reconstructed the patterns of gene activity...

...Background on neuronal tracing A key aspect to understanding the brain’s function is knowing its architecture...importantly, any connections the neurons make to other distant brain areas. It is desirable for tracing compounds...the direction of information flow in order to understand which brain regions communicate with each other...decades was essential to build up our current understanding of neuroanatomy and neural connectivity, providing...advantage of AAV vectors is that there are well-established protocols for their production and purification...expressed a fluorescent marker in areas close or distal to the injection site across the entire brain, ...connectome”, to be visualized in a uniform and standardized way (Oh et al., 2014). Images yielded from this...

...scientific titles generated from the United States Standard Occupational Classification System  and Wikipedia... in the United States (0.428). 4. The #1 (re)tweeted domain by scientists was Instagram Social media and...they are based on the US Occupational Employment Statistics (OES), but international Twitter users were included...don’t use Twitter, are you interested in getting started? Reply in the comments below or tweet us your reply.... Other Resources Check out the Top 50 Science Stars on Twitter Want suggestions on how to use social...

...Rab11, and Rab35 to understand their roles in forming the cilia that help establish left-right organization... and limitations – for example, microtubule-destabilizing drugs are impossible to target to specific subcellular...available as lentiviral plasmids if you want to make a stable cell line. This design allows highly localized,...reversible microtubule severing and has very low dark-state activity and toxicity. All of that means this light-powered...dissect the roles of three Rab proteins during a key stage of zebrafish development (Aljiboury et al., 2023...After 30 minutes of blue light illumination, substantial PA accumulates in the plasma membranes, indicated...moderately-enhanced 10x and 30x SuperPLDs avoid dark-state activity and cytotoxicity better than the 50x and...

...suspenseful. 4. Start preparing your talk early - like today Don’t wait until you need to speak to start creating...’s attention will start to wander when you are supposed to be done. They will start looking at their phones...future boss in the room, so it is a good idea to start thinking about public speaking early and often. ... audience is that it can help you avoid making mistakes. I once made a joke about social science to an...Describe how you pushed to a solution or overcame obstacles. Take time to consider the order of your narrative...t be hard on yourself if you stumble or make a mistake. Move on, the audience doesn’t really care about...

...how CRISPR can take on HIV-1 in vitro, and what obstacles must be overcome for clinical success. ART and...ART-induced viral control found in most HIV-1 patients. Obstacles for CRISPR HIV-1 therapy Both of these methods...survive, but preventing re-infection would require sustained expression of Cas9/gRNAs, which could result in...enough T-cells could be reached to ablate a well-established viral reservoir. A second challenge for CRISPR...binding. It’s also possible that HIV-1 may evolve resistance to CRISPR therapies through PAM or seed sequence...multiple drugs to lower the odds of developing resistance. Shortly after Kaminski et al. published their...not ablate viral function, but rather promote resistance to CRISPR/Cas9. While these results are discouraging...

...to put together DNA fragments. As a fun way to start, I highly recommend watching this entertaining video...vector of choice as shown in the figure below. To start, you need to have DNA fragments with regions of ...not work well if the ends of the fragments have stable single stranded DNA secondary structure, such as...Gibson assembly isn’t ideal for synthetic biology standards, which rely up on the re-use of parts between ...One way around this is to use a combination of standard sequences with overlap regions, such as in the...to cleave the 22kb vector. When followed by the standard Gibson assembly techniques described above, this... to get mixed up with Gibson assembly (they all start with a G!) but actually work by significantly different...

...antibiotic resistance cassette and you aren’t introducing an additional antibiotic resistance cassette ...where the inserted DNA includes an antibiotic resistance marker. After transformation, plate the cells...into the plasmid backbone contains a kanamycin resistance cassette, you would plate the cells on kanamycin-containing...cells carrying plasmids that contain a kanamycin resistance cassette will grow. However, often times, the...often used for cloning in yeast since antibiotic resistance spontaneously arises in yeast. For example, Markus...occurring genes for cloning purposes (antibiotic resistance genes, toxin genes, and the sacB gene). By tapping...