...in vivo: acoustic reporter genes’ on our blog. Creative approaches to presenting the microbial world ...bacteria in the lab, I was excited to learn about creative approaches to visualize bacteria in an educational...educational setting. The Symposium on ‘Creative Approaches to Presenting the Microbial World’ showcased complex... comics (Dr. Sanja Saftic) to educational gifs created by the Amoeba Sisters. Here’s one example of how...lactose-around-we-can-make-some-enzymes-for   The take home message was that being creative and telling a story makes scientific topics both... fascinating to see the living art designed by creative minds with a (I assume) very steady hand. By connecting...connecting art and science, a fascinating interface is created that produces loads of inspiration for the current...

...genome-wide screens, introducing bias into all work based on these and any other CRISPR screens using the..., race. Many researchers do large-scale CRISPR screens in cancer cell lines, looking to uncover genetic...targets and related biomarkers . Data from such screens are often uploaded to the Cancer Dependency Map...Boehm labs pulled data from more than 1000 CRISPR screens in DepMap to see if they could identify the ancestry... American, Africa, or South Asian) used in the screens based on the cells’ germline genetic variations...about 90% of the cell lines used in DepMap CRISPR screens are of either European or East Asian ancestry, ...actually reference?  The CRISPR guides used in the screens they analyzed were designed to have a perfect match...

...breaks caused by Cas12 create short 3’ overhangs, which leads to slightly increased efficiency when conducting...type to subtype to variant (shown in italics). Created with BioRender.com.   Class 1 versus class ...underutilized compared to Class 2. Class 1 Despite their incredible abundance, Class 1 CRISPR systems are less popular...Cascade. As you might imagine, this is handy for creating large genomic deletions. Type I systems are also...with Cas3 (pink). Complex shown is from type I-E. Created with BioRender.com.   Type III Type III is ...most engineered Cas enzyme, with adaptations for increased fidelity, activation/repression, high precision...CRISPR-Cas9 mechanism, representing type II-A. Created with BioRender.com.   Type V Type V uses Cas12...

...and Carrot, and further means to increase aptamer stability and decrease background in vivo. The nature ...enrichment (SELEX), some 1010 oligonucleotides could be screened for their affinity to a wide range of non-nucleotide... visualization techniques. In addition, their incredible versatility opens aptamers for use in many entirely...aptabeacons (or aptatags) were born. Aptabeacons can be created in many different ways, but the general premise...that bound the compound malachite green (MG) to create a non-protein based visualization technique. Ultimately... knowledge of the nature of GFP fluorescence to create an improved fluorescent aptamer system. Knowing... RNA aptamer that would bind the fluorogen and increase its fluorescence, essentially mimicking GFP.  ...

...reactions. The BP reaction creates an attL-flanked entry clone. The LR reaction creates an expression clone ...reactions. Recombination of attP and attB sites creates attL and attR sites.   Gateway vectors contain...Gateway plasmids.    Figure 3: Method A to create an entry clone: recombination of an attB-flanked...attL-containing entry vector.   Figure 4: Method B to create an entry clone: TOPO cloning the insert into an...attL-containing entry vector.   Figure 5: Method C to create an entry clone: Restriction cloning the inset into...vector will recombine with the attL-entry clone to create the expression clone.    Figure 6: Generating...between the entry clone and destination vector creates two products: the desires expression clone and ...

... progress on the NSF “G to P grand challenge.” Creating a productive, new scientific community is no small...in particular, that they all share the plasmids created under this programs via Addgene. New organisms ... organisms) -  perhaps you’ll choose to study Crepidula atrasolea or Prochlorococcus with your next project...crisis. I like how they are using protocols.io to create a protocol community! Asclepias syriaca and Asclepias...used to study aspects of cognition. Spiralians - Crepidula atrasolea (Jonathan Henry) This black footed slipper...can express fluorescent proteins. This lab has created an automated tank for feeding and maintenance that...real bottleneck. New gif for #MolluscMonday: #Crepidula atrasolea juvenile, light microscope with polarizing...

... we grew. As the number of deposited plasmids increased, the accompanying sequencing data collection and...bioinformatics workflow management system As novices to creating a robust data pipeline that would be reliable ...community of Nextflow supporters existed who had created a shared resource of pipelines for genomic data... with others without any complicated setup. We created, tested, and debugged each of our pipeline steps...running the pipeline locally and in the cloud. Creating a bioinformatics workflow in Nextflow Establishing...scripting language that can be executed on Linux, we created our first process by simply copying and pasting...was working as expected, we moved forward with creating the next process and linking the output from the...

...vesicles with nonlinear contrast. Increasing expression and decreasing genetic footprint We took a cue ...heterologously by bacteria inside the body. So we screened gas vesicles gene clusters from a diverse set ...expression heterogeneity required single cell screening to isolate a clonal cell line capable of producing... expression to appreciable levels. We wanted to create second-generation mARGs with higher expression,...reduced the number of copies of gvpA needed to increase expression. We were able to express Anabaena ...expression leads to greater amount of gas vesicles and increased ultrasound contrast. In all the cell lines we ...their tracking with ultrasound imaging (Fig. 2d). Creating a cell line can be a time-intensive endeavor. ...

...polymerase, 2) increased PCR extension time, 3) increased concentration of MgCl2, 4) increased concentration...PCR reaction is created by making some or all of the following modifications: 1) increased concentration...saturation mutagenesis Several methods can be used to create mutational scanning libraries. While each of these...Therefore, Gibson assembly is better suited for creating small, focused scanning libraries. PFunkel:...library that’s ready to be tested in a functional screen of your liking. This method is similar to site-directed... screen...

...was identified from a high-throughput screen, selected for increased voltage response, brightness, and photostability... They use a unique and powerful high-throughput screen to select variants based on performance metrics...resulting JEDI-2P has faster response kinetics and increased brightness, sensitivity, and photostability under... be generalized to other RTKs of interest, they create sensors for FGFR1, EGFR and ErbB2. And orthogonal...developed using a high-throughput multiparameter screen (Koveal et al. 2022). Lactate is a key metabolite...cellular concentrations of lactate. The microfluidic screening platform developed here is also a promising approach...Figure 6: A) Schematic of BeadScan platform for screening candidate biosensor variants for performance under...

...as a stepwise decrease in signal strength in a flow plot. d = days post treatment. Created using biorender.com...binding and (b) antibody host species interactions. Created using biorender.com. Another way of indirect...1b). As above, the advantage of this system is increased flexibility for panel design. Another perk is ...can be bound by several secondary antibodies, increasing the fluorescence signal intensity. However, you...cell, CD4+ T cell, dendritic cell, CD8+ T cell. Created using biorender.com.   Intracellular labeling...cytosolic proteins, as well as proteins of the secretory pathway. Those can include cytokines and chemokines...bind to their targets in the cytosol and nucleus. Created using biorender.com.   Dye stains Dye stains...

...greatly reduced costs per plasmid. We generally create kits to allow you to easily use a set of plasmids...markers built into all plasmids in the kit help decrease false positives observed in the cloning process...protein combinatorial library by in vivo functional screen." Scientific reports 5 (2015). BIOFAB plasmid ...set 3000 Series Confronted with the problem of creating vectors for predictable protein expression in ...bacteria, researchers at the BIOFAB and the Endy Lab created this kit. The plasmids within it contain a variety...assembly. PhD student Dejan Gagoski used SITS to create the pCell-Free vectors, Gateway-compatible backbones...these different modular repeat segments, you can create a PUF protein that binds your RNA sequence of interest...

...topics discussed during the symposium will prove incredibly important to the stability of the biological ...align trainee expectations with job outcomes, and increase diversity in the academic research environment...these problems and some that stuck out to me were: Decrease the Number of Postdocs De-incentivize going into... more services based on what they're spending. Increase the number of science masters programs to prepare...postdoc salaries as opposed to Staff Scientists Increase the Starting Salary for Postdocs to $50,000 (the...salary as reported by Paula Stephan is $40,000) Increase Knowledge about Postdoc and Graduate Student Career...was highlighted during the conference that just increasing funding for academic research will not solve ...

...payload to a cell but cannot create new particles. As a bonus, this increases the carrying capacity of the...non-enveloped virus (left) and an enveloped virus (right). Created using biorender.com   If you’re new to talking ... Figure 2: Baltimore replication classes. Image credit: Louten, 2016 Viruses with DNA genomes can either...After a virion infects a cell, the single particle creates many copies of itself, sometimes up to millions...the risk of infection — researchers were able to create far safer viral vectors that can be used in BSL... the viral vector, increasing both safety and usefulness of the tool. In fact, most gutted genomes have...have as much genome removed as possible, just to increase the viral vector’s carrying capacity. In order...

..., the Liu lab created and evaluated 19 PE1 variants with RT mutations known to increase activity, enhance... initial nick site (to avoid creating a double strand break), increasing editing efficiencies 2-3-fold... Figure 1: An overview of prime editing. Created with BioRender.com. How does it work? First... between the template and primer binding site, increase processivity, or improve thermostability. What...fold. However, the process of double nicking increases indel formation slightly. Designing the sgRNA with...inventors kept optimizing it. Using a pooled CRISPRi screen, the Liu lab discovered that cellular mismatch ...addresses the problem of RNA stability. Using a CRISPRi screen, they identified a protein that enhances prime ...

...physically joining multiple fragments of DNA to create a synthetically designed DNA sequence. There are...fragment joining, but this is fine for its goal of creating a choice of standardized constructs and tools ... regions. Golden Gate Assembly also allows the creation of standardized DNA constructs, but its use of... expediently join multiple synthetic fragments, create multiple mutations in one or multiple fragments...well-designed fragment overlap sections, while still creating scarless plasmid inserts.  Want to Learn More...and eliminates the annealing step usually used to create a double-stranded oligo. Generating an sgRNA construct...directly transformed into E.coli for plating, screening and generation of sgRNA. Generation of sgRNA libraries...

...lower its aggregation tendency, increase its brightness, and decrease the amount of light required for...Fluorescent proteins have enabled scientists to pursue creative research avenues previously unavailable to them...applicants but Xin and Fatima stood out for their creativity in the use of fluorescent proteins and the potential...Fluorescent proteins can be applied in a long list of creative ways. In her graduate work in Michael Lin’s lab...by using photoswitchable fluorescent proteins to create enzymes that can be turned on and off with light...researchers will be able to use similar designs to create a wide variety of enzymes and proteins with inducible... to produce HMOs, sensor binding results in an increase in fluorescence of cells that actively produce...

...site-saturation mutagenesis, and medium-throughput screening, she increased the affinity of the sensor for serotonin...overwhelming response and were excited to see the many creative uses of fluorescent proteins. We’re happy to announce...thousands of protein variants simultaneously  With increasing sequencing power, scientists are able to quickly... protein-coding pathogenic human variants are decreased in protein abundance, possibly due to misfolding...neuromodulator, and evidence suggests that it is incredibly important for nearly every aspect of life.” Unger...system. “This bottom-up approach will allow me to create a fully controllable and reproducible set up,” ...explains. Developing an artificial leaf replica means creating a “leaf” with similar permeability of water and...

... primary screen to knowing what are the real hits is so much shorter…. it’s a great screening technology...come here, I need to spend time here, this is an incredible place.” Doench has spent most of his research...researchers in a variety of fields develop RNAi screens to answer their own research questions. Starting... For his post-doc, Doench worked on using RNAi screens to learn more about cancer biology in Ed Harlow...developing CRISPR pooled libraries for use in genetic screens and has contributed much of his knowledge to the...was completely justified… it has been just an incredibly useful technology.” He goes on to explain that...become more efficient in terms of editing and screening) such that thousands of individual edits can be...

...fusing miniTurboID with dCas9 to create TurboCas, the Shilatifard Lab created a very quick and efficient way...find in this post: Glutamate indicators with increased sensitivity and tailored deactivation rates Addgene's...targeting interneurons Glutamate indicators with increased sensitivity and tailored deactivation rates By...2025).In this latest work, the team designed and screened a large library of iGluSnFR3 mutants, identifying...Podgorski, K. (2025). Glutamate indicators with increased sensitivity and tailored deactivation rates. bioRxiv...puromycin resistance gene makes stable cell line creation a breeze. Modular by design: Key elements (promoter...protocols for viral generation and stable cell line creation (Figure 2). Looking for more? Explore our empty...

...this approach can greatly increase the sensitivity of an assay by increasing the number of signaling molecules...green; and proteins are represented in orange. Created with BioRender.com.    How do secondary antibodies...shorter than that of the primary antibody, due to increased binding affinity, many indirect antibody protocols...are designed to take place over multiple days. Increased run time is considered one of the major drawbacks...specific isotype subclasses of primary antibodies can increase the possible number of target proteins.   The ...immunohistochemistry advances have found ways to increase signals from conjugated primary antibodies. The...as nanobodies, has introduced new options for increasing signal strength.  For the time being, though,...

... the untagged allele. Created with BioRender.com. The first step in creating the plasmids was to identify...be prohibitively expensive for many labs. We’ve created and tested plasmids that use CRISPR/Cas9 to endogenously...first exon of the gene. In this manner, we have created an initial set of 10 plasmids representing 10 human...linker, and the GFP tag sequence to be inserted. Created with BioRender.com. A key feature of our methodology...the double strand break and introducing the tag. Created with BioRender.com. Plasmids, plasmids everywhere...strategy and design described above can be used to create similar donor plasmids for introducing tags into...

... older plates could increase plasmid loss or mutation. Let it incubate Increasing the incubation time ...prep We'll discuss the following techniques to increase plasmid yield. Keep in mind that you might need...particularly stubborn prep. Check the bacterial strain Increase the volume Improve the culture media Let it incubate...more extensive listing of common E. coli strains. Increase the volume Starting with a larger culture volume...you are working with a low copy plasmid. When increasing the culture volume, be aware that a higher volume...origins of replication (e.g. p15A, ColE1) can be increased by adding chloramphenicol to the culture medium...glycerol or glucose (in moderation) may also serve to increase cell density and plasmid yield. Start with a single...

...Laws Bring Increased Postdoc Wages Our most popular blog post this month shares incredibly important information...enacted labor laws will likely increase postdoc earnings in the U.S. This increase, while overdue in my opinion...considering a postdoc. In “Changing Labor Laws Bring Increased Postdoc Wages,” Future of Research executive director...variety of downstream effects including possibly decreasing the number of postdocs various academic institutions...Science Communication Changing Labor Laws Bring Increased Postdoc Wages I hope you all have a great November...

...experiment. Here, we will provide an overview of how to create and use a standard curve and provide some general... I make the standards for a standard curve? You create a standard curve by serially diluting a known control...mix the samples well by vortexing or inversion. Created with BioRender.com.   When choosing a standard... vortexing between each step of the series. To increase the accuracy of the curve, avoid pipetting small...value. How do I plot and use a standard curve? To create a standard curve, you graph the measured response...wider span of concentrations but suffers from decreased specificity, especially as it approaches the minimum... all of the samples in the assay, as this will increase the accuracy of the data. For assays involving...

...transdifferentiated in vivo to pancreatic β-like-cells with the expression of pancreatic and duodenal homoeobox... [13]:  Germ cells [14] Hepatocytes [15] Pancreatic β-cells [16] Intestinal tissue [17] Lung epithelial...rare in mammals, an example is observed in the pancreas when excess β-cell damage results in the transdifferentiation...cell types such as fibroblasts, hepatocytes, and pancreatic exocrine cells have been successfully transdifferentiated...differentiation of human ES cells and iPS cells into mature pancreatic insulin-producing cells. Cell Res, 2009. 19(4.... 38. Thorel, F., et al., Conversion of adult pancreatic alpha-cells to beta-cells after extreme beta-...PubMed PMID: 3690668. 40. Ferber, S., et al., Pancreatic and duodenal homeobox gene 1 induces expression...

...enable the creation of CAR-T cells directly within the body. Overall, DIRECTED can increase on-target ...reprogram delivery vehicles like lentiviral vectors, CreVLPs, and eVLPs for in vivo and in vitro applications.... The Ting lab recently made two deposits that increase the utility of this technique.  By engineering... O’Neill The Cohen lab and colleagues recently created a system for recording the transcriptional history...resembling a “ticker tape” recording. The authors created externally-timed bands of color with HaloTag-iPAK4...thin sections of tissue, the Wu Lab is working to create a three-dimensional atlas of protein expression...-95 [K28/43R] (70 µm mounted sagittal section). Credit: Zhuhao Wu, Weill Cornell Medicine.  Interested...

...Jim Woodgett. Public Library of Science (PLOS) created a stir earlier this year when it announced its ...manuscript submission. The rationale includes increasing the ability of reviewers (and, once published...some cases, typically consortia involving genomic screens, the data stream is so prolific that raw information...require access to each individual video. To PLOS’s credit, the organization subsequently clarified what the...and this alone is a reasonable motivation for increasing both data accessibility and methodological detail...Minimally, raw but sufficiently annotated data used to create charts and tables in the paper are in scope. For... up new opportunities for research as well as increasing transparency. Thank you to our guest blogger...

... to each to make the library usable for pooled screens. Applications of the cancer pathways ORFs kit Wood...and his colleagues developed the kit for use in screening for pathways that drive resistance to drugs in...the only way to use the kit. “You could use it to screen for pathways that confer any phenotype of interest...drug resistance,” he said. “It could [be used to screen for] morphological features, migration, or survival...use our libraries to do things - sometimes drug screens and sometimes totally different experiments we ...Tracing Read How CRISPR Can Be Used in Genome-Wide Screening Browse 28 Hot Plasmid Technologies from 2015 ... cancer screening...

...developing zebrafish pancreas. They first tagged these cells using plasmids. Then using HOTcre, a method that...of quiescent and proliferative pancreatic β-cells identified by HOTcre mediated labeling." Proceedings...these systems check out Gerald Rubin’s lab who created a set of modular vectors that can be used to optimally...unicellular organisms, they can also be used to easily screen multiple therapeutic compounds in one experiment...embryos with DNA or RNA to edit their genomes or to create transgenic animals. Zebrafish are an ideal model...β-cells that originate in different regions of the pancreas and produce different levels of insulin (Hesselson...

...Creating great educational science videos is tough. Is the content accurate? Are we conveying complex...people in tight locations. Conference rooms for on-screen presenters! Labs and benches! Everything was out...videos. It’s really nice to have a scientist on-screen, somebody who really knows the protocol and can...would be to use an iPad to shoot.  Build: Jennifer created a mobile camera area that included iPad and her...where I could focus on camera and audio, and the on-screen talent could just focus on their interview or performance...re-opened very early in the pandemic with some incredibly strict safety protocols to keep Addgenies safe...camera) would remain masked, it freed us up to be creative.  Test: Our first official shoot went off with...

...Symptoms of AIDS include rapid weight loss and an increased risk of infection, including both common infections...ART-treated patients lack plasma HIV-1, they are at increased risk for other chronic diseases, including dementia...disorders, nerve damage, and cardiac disease. This increased disease risk is attributed to latent HIV reservoirs... used histone deacetylase (HDAC) inhibitors to increase transcription of the integrated HIV-1 genome. ... enhancer region near the NF-KB binding sites increased activation of HIV promoter-luciferase constructs...microglial line, treatment with CRISPR/Cas9 SAM increased the percentage of cells with activated HIV-1, ...cell. ZFNs and CRISPR/Cas9 have also been used to create mutations in genes essential to HIV-1 replication...

...scaffold. To increase expression, Tanenbaum et al. modified the GCN4 sequence to increase its alpha-helical...activation. Multiple nuclear localization signals (NLS) increase protein import into the nucleus. In developing... Weissman labs took this biological lesson and created a system to amplify fluorescent signals. Named ...protein is expressed at low levels, you have to increase your imaging time to get enough signal. This workaround...cells without affecting protein function, thereby creating a single-molecule reporter of intracellular processes...alpha-helical structure and stability, creating v4 SunTag. Since the v4 system does not display protein aggregation...

...resulting in decreased BRET2 signal. With increased concentration of drug, the BRET2 signal decreases. ...leads to downstream effects.  Bryan Roth’s lab created a collection of plasmids called TRUPATH to interrogate...the complex will disassociate, resulting in a decreased GFP2 emission. The activation state can be measured...showing the effectiveness of using TRUPATH as a screening technique. Lastly, the activity of both antagonists...plasmids are available as a collection, facilitating screening across the transducerome, to gain a more complete...

... Figure 1: Initial steps in the CRISPR-pooled screening using sgRNAiBAR. Image from Zhu et al., 2019.  ... Peking University has developed a new CRISPR screening method that makes use of guide RNAs with internal...false-positive rate discovery rate, conventional CRISPR screens require a low multiplicity of infection (MOI) during... annotated human gene, it enables genome-wide screening with a high MOI, which is beneficial for a low... calcineurin dynamics and can serve as a drug-screening platform to identify new antifungals that target... assay, and are not suited to high throughput screening, calcineurin dynamics measurements, or monitoring... attached fluorescent protein to the DNA. They created plasmids for purification of tTALE-eGFP and tTALE-mCherry...

...finding. Transcriptional program changes from this screen successfully differentiated cells resembling all...from Joung et al.)    Putting MORF to work Screens and selections In its current format, the MORF ...library is most immediately useful for TF library screens. Previously engineered TF libraries were not as... library can be used with other selection and screening mechanisms, although the unique barcodes for each...mutation and/or TF dysregulation. A targeted TF screening approach can not only lead to a better understanding...better simulate a disease in a model system. TF screens can be designed to identify custom TF programs ...Cas-9 knockout and transcriptional activation screening. Nat Protoc. 2017 (4):828-863. doi: 10.1038/nprot...

... complexity of the device increases, as does the price. With this increased complexity comes the need ...adapted and combined with creativity and feedback from the community to create state-of-the-art devices...via workshops, seminars, or hackathons greatly increases accessibility. Through this approach, the Planktoscope...Madagascar. Platforms that allow for on-going creativity with the construction plans. Appropriate open-source...sources within the next few years, with the goal of creating more transparent and reproducible science in the...

...approach, the Anna Cereseto Lab created pX-evoCas9, which they showed also increased specificity. Most recently...broader PAM specificity can target more sites. Created with BioRender.com. Specificity Specificity...multiple PAM sequences (NG, GAA, and GT), greatly increasing the number of genomic loci that can be targeted...developed SpCas9-HF1. Both of the enzymes showed decreased off-target activity relative to Cas9. Since then...accurate Cas9 (HypaCas9). This nuclease showed decreased or similar off-target activity, relative to eSpCas9..., et al. "Directed evolution of CRISPR-Cas9 to increase its specificity." Nature communications 9.1 (2018...

...new Antibody Guide and not just because I helped create it. But while antibodies, as the OG (as the kids...reagents we just didn't have the space to give proper credit to. Thankfully, as Addgene's resident blog editor...the majority of these alternative reagents are created in the lab, instead of generated in vivo, they ...Listed in no particular order.) Fab fragments are created through enzymatic cleavage of the variable region...in clinical applications. Fab fragments can be created in lab in situations where a suitable antibody ... fusion proteins similar to a Fab fragment but created through engineering instead of cleavage. They are...cheaper to make than antibodies. Minibodies are created by binding together two scFvs with a CH3 linker...

...antibodies produced from its serum several weeks later. Created with biorender.com. To generate polyclonal ...additional booster immunizations every 2-3 weeks to increase the antibody titer. The titer is tested following...epitopes of the target protein. While this can increase background staining (see next section), it may...experimental protocol, using a polyclonal antibody increases the likelihood that one of the clones will still...crosslinking (ChIP) or fixation (IHC). Because of their increased sensitivity, polyclonal antibodies are particularly...epitopes of their target protein thereby greatly increasing signal amplification.  When to stay away from...population recognizes many different epitopes, increasing the likelihood of cross-reactivity with other...