... at the nucleotide level but share an overall structure of 13 base pair palindromic repeats separated ...efficient The first version of FLP discovered has a temperature optimum of 30 °C and is therefore inefficient...led to the identification of FLPe which has a temperature optimum of 37 °C (Buchholz, Angrand, and Stewart...Visualizing single neurons in the living brain   Figure 1: Plasmid mix to label neuronal morphology (eYFP...transfection efficiency. However, if the cellular structure of a neuron and its synapse should be visualized...Combing FLP and Cre for additional control   Figure 2: Expression of a morphological marker (eYFP) ...FLP Recombinase Evolved by Cycling Mutagenesis.” Nature biotechnology 16(7): 657–62. PubMed PMID: 9661200...

...minutes to point me in the right direction for my future work. She graciously took me for coffee and spent...conference for any reason was a Singleton. Figure 1: The Binning Singletons logo. Image: James et...that binning Singletons together would provide a structure and support network for people attending large...improvement. We will make a greater effort in the future to match people within similar fields (ASM Microbe...that all participants would benefit from more structure with regard to the Mentor-Singleton relationship...addressing. Clear expectations have been established for future participants regarding the activities and time ...scientific community can improve the diversity and culture of science. We want Binning Singletons to be another...

... Staphylococcus aureus Cas9. Nature 520:186–191 . https://doi.org/10.1038/nature14299  Senís E, Fatouros...bacteriophage and plasmid DNA. Nature 468:67–71 . https://doi.org/10.1038/nature09523  Ibraheim R, Song C-Q, ...SI, Dohmae N, Ishitani R, Zhang F, Nureki O (2014) Crystal Structure of Cas9 in Complex with Guide RNA ... pool of shorter orthologs, only Staphylococcus aureus Cas9 (SaCas9, 1053 amino acids) displayed cleavage...a distinct family of RNA-guided genome editors. Nature 566:218–223 . https://doi.org/10.1038/s41586-019...

...automatically using motion capture software such as EthoVision. Consult the literature to find all the applications... is an approximation of reward or the sense of pleasure that the animal perceives through taking a drug...may affect the animal; stimulation could induce seizures or some other unwanted phenomenon or it could ... elegans [10]. If used properly, optogenetics is sure to teach us much in all of these species. Happy ... nucleus accumbens facilitates reward seeking." Nature 475.7356 (2011): 377-380. Pubmed PMID: 21716290...direct comparative analysis of microbial opsins." Nature methods 9.2 (2012): 159-172.5. Goshen I, et al...."An optogenetic toolbox designed for primates." Nature neuroscience 14.3 (2011): 387-397. PubMed PMID:...

...scientists was Nature. Overall scientists tweet URLs with scientific domains <10% of the time. Figure 3: Top ...by biologists were: Nature, Sciencemag, Wiley, Cell and Scientific American Figure 4: Top Scientific Domains...have the most Twitter users. Biologists rank #8. Figure 1: Highly Represented Scientific Disciplines on...scientists on Twitter are male and 40% are female Figure 2. Gender Ratio of Scientists on Twitter. 60% of...scientific domains more often than other scientists   Figure 5: Portion of Tweets with Scientific Domains, Categorized...their fields than scientists outside their fields Figure 6: Follower Network of Scientists on Twitter. Each...

...updated version of BRET called BRET2. Figure 1: TRUPATH measures heterotrimeric G protein dissociation...can be measured by a BRET2 ratio, which is the ratio of GFP2 emission to RLuc8 emission (see figure). What...? The TRUPATH collection allows researchers to measure the physical association of combinations of different... signaling pathways.  This dissociation can be measured using a technique known as Bioluminescence Resonance...Rluc8 was more challenging. The Roth lab used structure-guided protein engineering and experimental refinement...both antagonists and inverse-agonists could be measured using the TRUPATH system. Conclusions Although...

...post about workplace bullying featured on the always interesting Tenure, She Wrote blog. Unfortunately...little pre-work most PhD students and postdocs do to ensure the advisors they choose will be ones that help...choose a field that you may want to work on in your future career, so the research topic will be more important...scientists to change labs – do as much as you can to ensure a good fit in advance. When a lab is interviewing...interviewing the lab to find a good “fit” and to ensure the PI will be a good role model and teacher. Do...creates a level of familiarity and confidentiality to ensure more honest answers. Ask people who work in the... to speak to them more than once to get a full picture and listen for hints at the same concerns from ...

...Jaenisch, Nuclear reprogramming and pluripotency. Nature, 2006. 441(7097): p. 1061-7. PubMed PMID: 16810240...cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 2006. 126(4): p. 663...human induced pluripotent stem cells (iPSCs) by temperature-sensitive Sendai virus vectors. Proc Natl Acad...differentiation of human ES cells and iPS cells into mature pancreatic insulin-producing cells. Cell Res, 2009...pluripotent stem cells into intestinal tissue in vitro. Nature, 2011. 470(7332): p. 105-9. PubMed PMID: 21151107...Differentiation of human induced pluripotent stem cells to mature functional Purkinje neurons. Sci Rep, 2015. 5: ...Dedifferentiation, transdifferentiation, and reprogramming: future directions in regenerative medicine. Semin Reprod...

...methods termed proximity-based labeling to better capture these interactions in a vast array of biological... of biotin for avidin, proximity labelling can capture weak/transient protein interactions as well as ...exposed lysines on neighboring proteins. Thus to capture protein complexes, scientists can simply express...After a few hours biotinylated proteins can be captured on streptavidin affinity beads or matrices. The...labeling in living cells and organisms with TurboID." Nature biotechnology 36.9 (2018): 880. PubMed PMID: 30125270...for electron microscopy and proximity labeling." Nature methods 12.1 (2015): 51. PubMed PMID: 25419960....genetically encoded reporter for electron microscopy." Nature biotechnology 30.11 (2012): 1143. PubMed PMID: ...

...necessitating the development of a new technique. In a structure as tightly packed and interwoven as the brain,...previously visible with Brainbow-1 and -2. The general structure of Brainbow-1.0 is retained in Brainbow-3.0, but..., but can be detected via immunostaining. This feature facilitates screening of Cre-negative Brainbow ... H, Draft RW, Bennis RA, Sanes JR, Lichtman JW. Nature. 2007 Nov 1;450(7166):56-62. PubMed. A technicolour...the connectome. Lichtman JW, Livet J, Sanes JR. Nature Reviews Neuroscience. 2008 Jun;9(6):417-22. doi... P, McKellar CE, Hall D, Looger LL, Simpson JH. Nature Methods. 2011 Mar;8(3):253-9. doi: 10.1038/nmeth..., Alexandre C, Bell DM, Dickson BJ, Salecker I. Nature Methods. 2011 Mar;8(3):260-6. doi: 10.1038/nmeth...

..., resulting in significant background staining (Figure 1A). Not all tissues will have the same levels ...species on species staining with no problem! But to be sure that your secondary is not binding endogenous IgGs...following three strategies might help!     Figure 1: Species on species staining can be a problem... secondary binding Direct immunohistochemistry (Figure 2A)  How do you avoid having your secondary antibody...Pre-formed primary and secondary antibody complexes (Figure 2B) Some labs have found that you can significantly...as your primary/sample, and then applying this mixture to your tissue (Tuson, et al., 1990; Goodpaster...within a problem.  Pre-block with Fab fragments (Figure 2C) Speaking of Fab fragments… Another strategy...

...from? Or why they are such a commonly used cell culture model? In this blog we will spill the beans on ...postdoc Frank Graham’s 293rd experiment, hence the nomenclature we use today: HEK-293. The exact origin of the...making it genetically female. HEKs are generally cultured as an adherent monolayer, though derivatives have...However, Frank Graham showed HEKs display many immature neuron-like qualities, which is inconsistent with...cell line. What the HEK is wrong with it?   All cultured human cell lines are susceptible to contamination...immortalized, but cell health can degrade over time in culture. This can affect factors that could skew research... etc.). Thus, it’s important to maintain short culture periods (less than 20 passages) to prevent these...

...calcineurin activity measurements in stressed or aging cell populations. Figure 3: Micrographs of...library Article contributed by Susanna Bachle Figure 1: Initial steps in the CRISPR-pooled screening...infection (MOI) during cell library construction to ensure that the majority of cells receive one gRNA. The...Read our SPARC blog post for more details. Figure 2: Dense (c), intermediate (d), and sparse (e) ...target. Calcineurin reporters allow scientists to measure calcineurin dynamics and can serve as a drug-screening...high throughput screening, calcineurin dynamics measurements, or monitoring in vivo. To overcome these technical...dye Article contributed by Jennifer Tsang Figure 4: Double stranded DNA bound by tTALE-FP with the...

...10.1089/hgtb.2019.110 Pacouret S, Bouzelha M, Shelke R, Andres-Mateos E, Xiao R, Maurer A, Mevel M, Turunen...quality control (QC) mantra is to do our utmost to ensure that scientists are receiving the exact materials...substrates for tagmentation-based library preparation procedures. This finding eliminates the double-stranding...time and cost for AAV genome sequencing.  Figure 1: The viral genome sequencing workflow. Using... that looks for pre-defined serotype specific signature sequences in the data. Unlike previously described...not require any additional reagents or lab work (Pacouret, 2017). Moreover, the sequencing data from multiple...t new to Addgene, we are excited to share the procedures and Python scripts with laboratories and viral...

...many iconic papers, including the ones on the structure of the DNA, were not peer-reviewed (Baldwin, 2015...the study to be replicated or omission from the literature. I think that delivering constructive critique.... The general representation of data is... The figures are ... In general, I think that this is an (e....checklist for individual sections of the paper (see figures). After finishing my section-specific comments,...WormBase, Arabidopsis.info). These repositories will ensure that the results of your colleague’s hard work ...comments will learn from them and propagate this culture further.  Many thanks to our guest blogger Magdalena...salt-stress induced responses in Root System Architecture of Arabidopsis HapMap population in Amsterdam...

...affected by environmental conditions (such as temperature, pH, fusion protein, etc...).  There are many... making such a decision remains spread across literature in publications that introduce these tools. ...references, etc...) and a more robust database structure was needed.  It is also difficult to scale such...submissions also require a DOI referencing primary literature, and every effort is made to moderate and cross-check...cross-check all data with published literature as soon as possible after submission. Using FPbase The most...’ll find matching FP suggestions. If you’re not sure exactly what you’re looking for, the Table of Fluorescent...emission spectra when available, and will soon feature filter spectra and collection efficiency calculations...

...rough structure of the plasmid based on the predicted sizes and organization of different features within...and temperature compatibility when digesting with more than one enzyme. Consult the manufacturer’s manual...their defined recognition sequence. Most enzyme manufacturers make High Fidelity versions of the endonucleases...loading them. The glycerol in the buffer will make sure your sample settles in the gel well and the dyes...expected band sizes. Always run control uncut DNA to ensure your enzymes are working. When uncut plasmid DNA...approximately 75-80% of the way down the gel will ensure you keep smaller bands from running off; however...

...plasmid-based research using synthetic vectors and cultured mammalian cells. Of course, these mammalian vectors...Therefore, it is not always mission critical to make sure the plasmid replicates in the cell, as many of these...foreign material and will subsequently pass it on to future generations. Stably transfected cells may be considered...stable transfection, there should be a selective pressure to force cells to incorporate the plasmid DNA ... used in conjunction with positive selection to ensure your gene gets targeted to a specific location ...293HEK, Jurkat T cells Inhibits protein synthesis; premature chain termination 1-10 ug/mL Zeocin Sh bla HeLa...is often used as a supplement in mammalian cell culture to suppress bacterial growth, and is not appropriate...

... this class measures either factors as proxies for a cellular process or directly measure a cellular process...to the structure of actinin in low vs. high tension. These systems are powerful tools to measure real-time...boiled down pretty simply – they are systems that measure biological phenomena by generating an output which...microscope, plate reader, or any other instrument which measured GFP levels and converted that signal into a readable...will focus on those which can be applied in cell culture and animal models primarily. Fun fact: Almost all...tissue preps, in multiple species, and in cell culture models. Some of them have even been expanded for...

...passaged from an infected host-cell culture to an uninfected culture which results in less overall dilution... without DNA cleavage. Nature 551:464–471 . https://doi.org/10.1038/nature24644 Richter MF, Zhao KT, Eton...for speed: base editing before Cas lets go Figure 1: David Liu gives a talk at the Cell Symposia:...from the infected to a fresh uninfected host cell culture). This dilution step represents the major difference...dilution and less stringent phage selection. Figure 2: Phage-assisted evolution of base-editing activity...improved Cas domain compatibility and activity. Nature Biotechnology. https://doi.org/10.1038/s41587-020...