....” Subscribers to Scientific America would be considered the interested public and it would be safe to...scientific or science communication. A Tweet is still considered scientific communication if the language is aimed...  For example, while “PCR test” has recently transitioned from jargon to non-jargon in the context of ...of disease testing, it can still be considered jargon in most other uses. Many laypeople would use “PCR...Instead, it targets a specific audience - giving consistency, purpose, structure, and intent to the messaging...your audience, and their expectations of you, consistent and clear.   And now that we’re all on the same...

...aborted fetus. HEKs have an unusual ploidy and are considered hypotriploid: most chromosomes are represented...though derivatives have been adapted for growth in suspension. Of the many derivatives of HEK-293, the most...many immature neuron-like qualities, which is inconsistent with kidney cells (Shaw et al.). Since then,...systems, such as yeast. HEKs can also grow in suspension, which allows for large scalability, and can ...cells may be particularly useful due to their intrinsic properties. HEK-293 cells are tumorigenic, ...widely available, which can make for a seamless transition of biological tools to other researchers using...

...describe the discovery of a light-sensitive opsin from algae that is sensitive to red light instead of blue...The problem is that all of these molecules are sensitive to blue light. Many have tried to make green molecules...blue light molecule much, much, much more light-sensitive. Then you can use dim blue light and red light... tools in a deep part of the brain, making it sensitive to light. When they illuminated this region, the...for the first time? Boyden: There are always considerations for each species. This could be a three or ...

...Transferred DNA once inside the infected bacterium can either exist as transient extrachromosomal DNA,...inject their genetic material into the cell. Once inside, phages can follow one of two different life cycles...Lentiviral and AAV can be used to create both transient cell lines, where a gene of interest is expressed...Bacterial conjugation Conjugation was the first extensively studied method of gene transfer and was discovered...the other hand uses electrical pulses to create transient pores in the cell membrane that genetic material...

...Brightness Brightness refers to the fluorescence intensity of a fluorescent molecule. A higher brightness...sample. Practical brightness A protein with a high intrinsic brightness that does not fold well or cannot be...brightness and is of no use. Therefore, it is more sensible to compare fluorescent proteins based on their...wavelength, available emission filters, and detector sensitivity) and the biological system (temperature, prokaryote...brightness When a dish of mammalian cells is transiently transfected with a plasmid containing a fluorescent... there is huge variation in the fluorescence intensities of individual mammalian cells within the dish...

...” has me considering the microbiome more often than not). If you’re someone who’s considering getting ...definitely enjoyed the lab!) and would therefore consider myself a neuroscience novice (newb). In this post...’ve developed in your current work to make new insights in neuroscience. Even if you're currently a synthetic...altered function or maybe your favorite FRET-based biosensing technique can be applied to an important neuronal...experiments and to find tools that will make your transition into the field much easier! If there’s a plasmid...

...and tension all have specific sensors designed for them. For example, this class includes tension monitors..., which will turn on, change color, or change intensity of fluorescence in the presence of calcium. This...specific proteins of interest such as actinin. When tension is high vs. low in the cell the donor and acceptor...changes to the structure of actinin in low vs. high tension. These systems are powerful tools to measure real-time...reproducible when used as stable systems, rather than transiently overexpressing at the time of use. Another great...

...approaches can be found. As an example, say you are considering either a cellular screen or a selection. A selection...specific to your scientific question. Things to consider when selecting an assay system include: How specific...specific is it? Is it high throughput? Will it be sensitive enough? No one wants to spend time screening hits...t yield any hits. Make sure you plan ahead and consider these questions to save yourself a headache later...scene, there are other options which are worth considering. RNAi screens are similar to CRISPR, but instead...method, popularized in yeast, are temperature sensitive screens. These involve raising or lowering the...

...molecular switches in signal transduction pathways and consist of a trimer of three subunits: alpha (Gα), beta...function as one unit: Gβγ. In general, GPCRs show a propensity to associate with certain subtypes, although ...ligand-bound GPCRs of interest. The TRUPATH plasmid kit consists of 20 plasmids encoding 14 alpha, 2 beta, and ... internalization of two different receptors- angiotensin II type 1 receptor and the β2 adrenoceptor. ...ideal for chemogenetics as they are relatively insensitive to their endogenous ligand, acetylcholine, and...Pascolutti R, Paulo JA, Gygi SP, Kruse AC (2017) Multidimensional Tracking of GPCR Signaling via Peroxidase-...

...order to gain a competitive advantage this cycle, consider the advice below:  1. Eligibility check Scrutinize...and grades that serve as automated filters of consideration. If you are worried about any eligibility provision...which is the perfect opportunity to paint a comprehensive picture of your academic interests, potential... day of the interview, dress the part, and be consistent with your personal statement, while you make ...your close peers to come along for the ride, and consider applying to several opportunities. In an age of...success. Every student should use these guidelines consistently across a number of years as you apply for internships...

...with Addgene’s expansion into plant synthetic biology. There are already two comprehensive plant cloning... of the technology to the public in an open, responsible manner. At Revolution Bio, we are addressing ... circadian rhythm in petunias. Because of the extensive research that has come before us, it’s possible...change color. Without this basic research – this comprehensive understanding of the genetic pieces underpinning... intellectual property concerns – patents and licensing agreements. Developing a product without the use...

...school and turn it into a biotech company. Transitioning an idea from academia to a startup Mike Koeris... biotech startup, Mike explained, his career transitioned away from the bench and became all about finding...failure at all stages in the startup". To gain insight into "the random-walk" of a budding startup, I ...company won’t go anywhere. The team should initially consist of 2-3 individuals that are aligned in vision and...company may begin to prove the technology. This transition takes around 1 – 2 years. The product is the...into it.” Have any other helpful tips to help transition a project from academia to a biotech startup ...

...budding. Yeast Centromere plasmids (YCp): These are considered low copy vectors and incorporate part of an ARS...are most similar to bacterial plasmids and are considered “high copy”. A fragment from the 2 micron circle...spontaneously occuring resistant mutants and the insensitivity of yeast strains to some antibiotics. Auxotrophy...uses for said element. This link provides a more extensive list of yeast auxotrophic markers and includes...auxotrophic selection in E. coli? Additional considerations HIS3 L-hisitidine no S. cerevisiae  no yes...ade6+ purine (adenine)   S. pombe  no no   Considerations when using auxotrophic selection Of course,...

...interested in following? Someone you admire for their insight and way of expressing it in meetings? The more ...help you learn the local “ropes” and become an insider faster A mentor that works elsewhere is able to... If your organization has a mentoring program, consider tapping into it and helping make it work by being... coaches who charge for their advice and time. Consider this option but be cautious and make sure to choose... to be your mentor, it may sound like a big responsibility or commitment. Start by asking them to have...questions and topics to discuss. The mentee is responsible for leading the discussion and for being “coachable...

...recurrence, and treatment of cancer have been extensively explored. Biologists have attempted to study ...and dynamic phenomenon that is interestingly responsible for tumor reduction as well as progression. Dunn...is a region of incessant chemical activity, and consists of other cell types, especially immune cells and...relapse. Chemical and radiation therapies cause a transient shutdown of the immune system, hampering its ability...have been made to use whole cell therapeutics consisting of various types of immune cells. Dendritic cells...exhibit cytotoxic activity. CAR-T cells have consistently shown promise in reducing leukemia and lymphoma...impacted by the trial. During clinical trials, expansion of participants based on early results can enhance...

...thin plane within a relatively thicker three dimensional sample (see Figure 1); similar to an MRI or CT...rapid photobleaching and a reduction in signal intensity. 2-Photon microscopes restrict excitation (and...focal point of the objective can a a high enough density of red-photons be establish for this to happen....field of the objective (the distance in the axial dimension that will appear in focus). This means that the... then collected and projected back to a fast, sensitive detector such as a scientific CMOS camera. If ...Reversible Saturable Optical Linear Fluorescence Transitions (RESOLFT/STED) also excel on these types of samples...a host of tools and opened doors to many great insights, it can be overwhelming to a novice microscopist...

...in vivo, they have higher reproducibility and consistency in the final product. Because the constant (non-epitope...binding) region is the first thing to go when downsizing an affinity reagent, most of the alternative ...its thorns and every affinity reagent has its downsides. These reduced-in-size options often have reduced... as ankyrin repeat proteins. DARPins typically consist of 3-4 repeats, have high specificity and binding...it? This post isn’t meant to convert anyone to downsize their affinity reagents, trendy as that may sound...Grumezescu, A. M. (2018). Drug targeting and stimuli sensitive drug delivery systems. Elsevier Science & Technology...

...LoxP vector.   Turn it off for a little while Transient suppression of expression is faster and easier...molecules that degrade target mRNA transcripts. When transiently introduced, they can deplete endogenous protein...instead of introducing a DNA double strand break. Transient introduction of CRISPRi or inducible expression...expression. Addgene has a dCas9 KRAB plasmid for transient knockouts!  Turn it on Turn it on forever Permanently...activator and repressor mechanisms Want to transiently induce expression? Check out Addgene's Lentiviral...site-specifically knocked in. This can be labor intensive and time consuming. If your native protein doesn...

...experimental design considerations used in all behavioral studies. General considerations for behavioral ...behavioral testing As a general consideration for behavioral testing in a laboratory, a high level of experimental...increase stress levels and affect behavior. A fact to consider: Rodents can hear ultrasonic frequencies (> 20...targeted to a specific area. Some other technical considerations for stereotaxic injections are: Vector titer...brain areas, repeated staggered injections may be considered. Timing: High expression levels of the viral ...

...isotype choice. Target and application should be considered The first question you may have is whether a ...monoclonal primary antibody (A). To boost signal, consider using subclass specific secondary antibodies instead...immunoprecipitations. Your sample species should also be considered when choosing an antibody. In some applications...options are available to you, it may be worth considering an antibody raised in a different species.    ...      Table 1: Isotype considerations when choosing antibodies   Complexity is a key factor...with a distinctly conjugated secondary antibody. Consider too that some secondary antibodies can cross react...

...Your time is precious. Here are a few things to consider before you commit: Double check to make sure ...can be a particularly good resource if you are considering using a reagent for “off-label” purposes. When...about using Google for serious science questions, consider using Google Scholar, which searches scientific...email you’re writing is probably not directly responsible for the problem. We’re here to help. You've ... few days without getting a response, however, consider following up. Ask for a timeline if you are ...upfront how long it will take to get an answer, consider asking to avoid unrealistic expectations. And...

...details on how they work!) Figure 1: Base transitions are edits between the purine bases (adenine, ...limitation to this approach. CBEs and ABEs are base transition editors. They adjust the side groups of DNA bases...task, not a job for a single enzyme. Base transitions Base transversions C → TG → A A → GT → C ...some cases, the same repair pathways that were considered problematic in the development of CBEs and ABEs.... Like other ABEs, the editor they were using consisted of Cas9 fused to an engineered adenosine deaminase...rapidly. If it follows a similar path to base transition editing, we can expect several generations of...to repair DSBs. Base Editing terms   Base transition Base changes between purines (A ↔ G) or between...

...solution against a part of the body very sensitive to heat- the inside of the wrist or the cheek. (Parents ...work with, require extra serum to survive at low densities - adding to the expense of dilution cloning. ... transfected cells into 3 plates at different densities for selection). A 2% agarose solution is then ...plates containing selection agent at different densities, then dispose of the plates that do not appear...such as the Viaflo from Integra Biosciences) to transition from a 48-well cell plate to a 96-well assay ...

...class of FPs, the FP timers, add an entire new dimension to monitoring localization; using FP timers, researchers...production of dsRed E5. This timer predictably transitions from green to red fluorescence (500 to 580 nm...However, this initial timer was a tetramer with a propensity to aggregate in cells (Tsuboi et al.), limiting... in Xenopus embryos. Of course, timers may be sensitive to changes in oxygen, temperature, and pH, but...et al. developed monomeric FP timers with less propensity to aggregate. These monomeric FP timers (mK-GO... that differentiates human IPSCs into glucose-sensitive insulin-secreting beta-like cells." Nature communications...

...infects host cells through binding the human angiotensin converting enzyme 2 (ACE2) cell-surface receptor...receptor. The spike protein RBD responsible for binding ACE2 is the target of most anti-SARS-CoV-2 antibodies..., homogeneous time resolved fluorescence, and suspension bead assay (Figure 2). After finding no cross-reactivity...scFVs to cognate aaRS antigens as measured by suspension bead assay. Image from  Preger et al., 2020. ... a compact Cas9 ortholog from Staphylococcus lugdunensis that recognizes a NNGG PAM and has comparable...expressing GCaMP6f with this promoter. Image calcium transients in PV interneurons with AAV-S5E2-GCaMP6f are ...

...is its simplicity and versatility. CRISPR/Cas9 consists of two components: the non-specific endonuclease...libraries (referred to simply as “CRISPR libraries”) consist of a heterogeneous population of gRNA-containing... is right for you There are several factors to consider when selecting a CRISPR library for your experiments...identified using next-generation sequencing (E). Considerations and tips for successful screens Next-generation...check out Addgene’s 2016 publication “Practical Considerations for Using Pooled Lentiviral CRISPR Libraries...Waxmonsky NC, Swanson LE, Fan M (2016) Practical Considerations for Using Pooled Lentiviral CRISPR Libraries...

...As a person with many roles and responsibilities, I have to think a lot about how to balance those roles...meeting and a means to request funding for events, consider reaching out to it. PTAs have more flexibility...schooler himself and b) I had the background to be considered an authority in this area. What is genome engineering...presentation, she said “it was such a valuable extension to our curriculum and offered engaging examples...Educational Progress (NAEP), virtually none were considered proficient in science, regardless of race or ...posts Resources on Addgene.org Check out our comprehensive CRISPR guide Find CRISPR plasmids and resources...

... blog post discusses some of the key things to consider when performing colony PCR.   Designing colony...for the presence of many different inserts. The downside: this type of primer does not provide information...information about insert orientation, then you might consider designing orientation-specific primers. Blunt ...day, you can save the leftover bacteria-water suspension and use them to start cultures of your positive...up PCR reactions: The remaining bacteria-water suspension will serve as the template for your PCR reaction...tool to have in your molecular biology tool kit. Consider giving it a try next time you’re screening for...

...cells, thus expressing additional EBNA-1 transiently. The transiently expressed EBNA-1 increases protein expression...field [4]. Three major reprogramming methods are considered when generating hiPSCs for clinical purposes:...purposes: mRNA transfection- this allows transient expression of the reprogramming factors Sendai virus- a non-integrating...efficiently [5]. However, the Sendai virus method is expensive, especially if used to make clinical grade hiPSCs...OCT3/4 and shRNA against p53 pCXWB-EBNA1 37624 transient EBNA-1   Many thanks to our guest blogger,...

... elaborated on later. As for what job fairs to consider, some of the best options are the Naturejobs Career...most relevant experience at the top. You may also consider visiting any of the employer’s career pages to... apply for the position. This post on Business Insider nicely summarizes good job fair practice. Career...’t have a particular employer in mind, you may consider starting with indeed.com or monster.com, where... important to realize that you have to take responsibility for your own professional development, not ... Reading and Outside Resources http://www.businessinsider.com/how-to-get-the-most-out-of-job-fairs-2014...

...provenance (this is the history of the cell line consisting of records of the origin and propagation of the...cell line contamination: Epithelial Mesenchymal Transition Is Required for Acquisition of Anoikis Resistance... The same test is so good that it is used as a forensic technique (aka DNA fingerprinting) that can place...convince people of some of the basic tenets of responsible cell culture outlined in this article. Now many...studied biochemistry at Warwick University, gaining extensive cell culture research experience through working...for a range of testing, from animal doping to forensics, DNA sequencing to drug testing. You can find ...

...includes the aforementioned internal control and is consistent with the result in Figure 1 could look like this...associated with the independent variable should be consistent over time and from different preparations of ...replicate control is important, it speaks to the consistency of the reagents and hands performing the experiment...experiment, not the reproducibility or consistency of the observed phenotype associated with the independent...replicates control for the reproducibility and consistency of the observed phenotype associated with the...may be, proper controls are a basic tenet of responsible scientific inquiry and investigation. References...

...express our gene of interest. This element is responsible for initiating the transcription of your insert...eukaryotes have muliple polymerases which are each responsible for a specific subset of RNAs. To gain this specificity...transcript     Description Expression Additional considerations CMV General expression mRNA Strong mammalian...elongation factor 1 alpha Constitutive  Tends to give consistent expression regardless of cell type or physiology... used for Description Expression Additional considerations T7 in vitro transcription/ general expression...regulatable  Often paired with the temperature sensitive cI857 repressor. T3 in vitro transcription/general...

...these particularly energy-intensive systems in a more environmentally responsible manner.  Other ways to ...service items we employ dozens of pieces of energy-intensive lab equipment on a daily basis. By the beginning...100% renewable electricity! A (not so) shocking transition  In early July the Town of Watertown announced...substantially reduced in an attempt to save energy. The transition to 100% renewable electricity will allow us to...

...synthesized enzyme. Biotin protein ligase (BPL) is responsible for this highly specific post-translational protein...biotinylated. In E.coli for example, the BPL, BirA, is responsible for biotinylating only one lysine in the entire...Esmond E. Snell was able to purify the protein responsible for binding biotin, avidin. Avidin was aptly ...Kresege et al., 2004). Avidin is a glycoprotein that consists of four identical subunits that can each bind ...non-selectively label DNA and RNA. Photobiotin acetate consists of a biotin molecule attached to a photoreactive...Photobiotinylation is sometimes easier and less expensive than other enzymatic methods. Scientists have ...

...Faculty Position. This edition has substantial expansions on the chalk talks and Zoom interviews sections... and mentoring to several people that found my insights helpful. All went on to get faculty positions ...understand your proposal to give you proper consideration. 3. Present an easy to follow seminar See item...Implications for human disease" is vague. Instead, consider "A mechanistic understanding of this system will...free!) contains strategies, hints, anecdotes, and insights. After I wrote the first version of my manual,...publish this manual and earn some cash for my expensive habits of running shoes and single malt whiskies...

...being realized. There are a variety of reasons to consider using genome editing to change the genetic code...sequence and ~75 nucleotide scaffold sequence) is consistent between plants and other organisms, but the promoter...CRISPR components can be expressed stably or transiently depending on the delivery method and cell type...CRISPR components can be delivered and expressed transiently using a standard detergent, Polyethylene Glycol...GOI) into plant cells. A) The system typically consists of a Ti plasmid, into which the GOI is inserted...deliver CRISPR-Cas9 to plant cells and should be considered alternative to PEG or agrobacterium-mediated ...

...change on the opposite strand). Both are base transition editors: converting one purine base to the other...pyrimidine base to the other. Figure 1: Base transition editors catalyze purine-to-purine or pyrimidine-to-pyrimidine...BioRender.com. How does base editing work? Base transition editing requires three elements. They are, broadly... in David Liu’s lab in 2016. History of base transition editing Cytosine base editing The beginnings ...frequency of 1.2%. Although the lab did not conduct comprehensive genome-wide studies of ABE specificity, their... a similar approach. Dali Li’s lab’s A&C-BEmax consists of a fusion of both cytidine and adenosine deaminases...The other 8 base-to-base changes require Base Transition Editors, which will be discussed in another post...

... concern, or nothing to worry about all. Let’s consider several scenarios below for a CRISPR experiment...An off-target rate of 5% is far too high when considering human patients and the diseases associated with...many more. While such software typically only considers DNA sequence and doesn’t take into account chromatin...the genome to reduce off-targets, it is time to consider your Cas enzyme. Even if there are similar sequences...unnecessary for many applications, it is the only comprehensive method available. Targeted sequencing approaches...must be quantified slightly differently. For a comprehensive approach, RNA-seq would be an appropriate method...

...individual neurons, including dendritic and axonal extensions and, most importantly, any connections the neurons...neuroanatomy and neural connectivity, providing valuable insights for neuronal communication. Viral vectors and...synaptic step, thus preventing further widespread expansion of RABV. This improvement resulted in a clearer... Not all measures of transport efficiency are consistent which may be caused by the diversity of techniques...the field of AAV vectors. Conclusion An ideal comprehensive toolset for neuronal tracing and network identification... wheat germ agglutinin (WGA) as a new, highly sensitive retrograde tracer in the rat brain hippocampal...nucleus Rabies and neuronal tracing Important considerations when using AAVs Resources on Addgene.org ...

...someones) taking on these responsibilities on a team to which you belong, consider stepping up. Otherwise... function. When a select group of people are responsible for a group of specific processes, they must ...tasks done. At Addgene, the Scientist Team is responsible for guiding new deposits into the repository,...roles they’re meant to fill and what their responsibilities are. That’s a subtle but important distinction...- and really can’t - be done alone. So, if you consider yourself part of the scientific community, get...

...science policy community is the one that was consistently welcoming. This bears repeating. Individuals... early career researchers who have decided to transition out of academia from graduate school or a postdoc... came to DC to do either a sabbatical year or transition away from academia as tenured professors. There...Day One Project for some example proposals and consider applying for their accelerator for some hands-... to future positions, but will also make your transition from academia to the policy world much smoother...smoother.  Action Items:  Explore this comprehensive resource developed by Dr. Kelly Singel which provides...

... This post will go over some of the general considerations you should review before using an antibody....necessarily mean that you yourself have to perform extensive validation experiments for every antibody that...data can be acceptable. However, it is your responsibility to determine what counts as adequate validation...methods. These approaches have relatively low sensitivity, which can be problematic if you are trying to... your antibody’s trustworthiness. You may also consider sharing your validation attempts by other means... test an antibody (perhaps one from Addgene!), consider sharing your results so that others in the community...

...multiple high-profile publications. My classmates consisted of graduate students, post-grads, and early career...topic by the moderators. The assignments, which consisted of reading that class’s section of a manuscript...given the opportunity to sign up for a writing intensive weekend in Washington D.C. at the ASM headquarters...Four ways to improve your next manuscript 1. Be consistent with your figures The figures of a manuscript...and they should also be easily understood and consistent in design and layout. 2. Be concise in your writing...

...aspects of the subject you're writing and then condensing that and making it accessible to students.” Making...for example, cutting open a heart to look at the inside. We do a lot of interactives and videos, as well...specimens.” And since the eScience warehouse is onsite, Caitlin can visit the operations team to make ...curriculum, but has taken on other new and exciting responsibilities.“I’d say maybe 25% of my day is still what...lab: write!” Caitlin did this by editing 15+ comprehensive exam proposals, a few PhD theses, and occasionally...

...will discuss some of the features you may want to consider as you make your choice. The first and most important...selection marker or antibiotic resistance. Next, consider the expression level of your construct: would ...pcDNA3-EGFP). If you can get away with generating transiently transfected cells, you will not need a mammalian... lentiviral or a retroviral backbone. This is considered to be a more robust expression system and has...are several types of yeast expression vectors. Consider your applications and experimental design. You...

...society 6)   Consider participating in a future SciFund Challenge. While the goal is ostensibly to get some...magazines or newspapers or other places online, and consider how those stories are being told. Twitter can ...science blogs written by practicing scientists. 3)   Consider starting up your own twitter feed or talking about... both your plasmids and your research stories. Consider practicing communicating your research by guest...

...know all about antibiotic resistance genes, let’s consider another basic element of any plasmid: the origin... intend to use, and whether or not you need to consider your plasmid's compatibility with one or more ...copy number varies. See below for additional considerations. *Requires pir gene for replication (reference... external factors contribute as well. These considerations are especially useful to keep in mind if you...affect copy number. Some ori's are temperature sensitive; others ori's can be "tricked" into amplifying...

...University of North Carolina (plasmids at Addgene). It consists of two domains, LOV2 and Zdk1, where Zdk1 binds...attract other proteins involved in their growth and extension. Now, they split EB1 into two halves (Fig. 1):...RNA localization in mammalian cells. These tools consist of cobalamin-binding riboswitches that bind to ...single base-editors has resulted in the rapid expansion of the genome editing toolkit. However, a challenge...quantitative editing assay. To help accelerate the expansion of genome editing tools, the Reuben Harris lab...

...The light intensity used to activate the PA-FP should also be considered. If the light intensity needed ...your experiment, there are several parameters to consider, such as brightness, photostability, pH stability...protein aggregation. Another important parameter to consider is the contrast ratio- a comparison of PA-FP brightness...microscope will have it’s own quirks (level of zoom, intensity, possible excitation lasers) it is also important...