...done.   Figure 1: DNA melting by DnaA and IHF is the first step of replication for the bacterial chromosome...plasmids rely on their host cell's replication machinery in order to replicate—but not always to the same extent...described in our previous Origin of Replication post, DNA replication is initiated at the ori and may or...consider the replication mechanism of plasmids under stringent control, such as pSC101. DNA replication and regulation...review how E. coli normally replicate their chromosomal DNA. Chromosomal replication in bacteria is carefully...entire round of replication. Initiation is the primary control point of DNA replication in bacteria, so... the replication forks reach each other on the opposite end of the chromosome, ending replication.   ...

...first foray into Reddit. Here are some reasons why: 1. Reddit has a large and active community of people...re more interested in selling a product than communicating your ideas with people or discussing science...to write about in the future. The AMA was also cataloged for permanent reference by the Winnower. The only...I do a lot of public speaking where I enjoy communicating face to face with the audience. I post on LinkedIn...own expertise and your resources for science communication opportunities like an AMA. One of the best things... science communication...

... and, if you do it right, can be (part of) figure 1 of your next paper or thesis. This series of posts...An explanation of the assay is depicted in figure 1. The strict correlation between the fluorescent protein...protein for your application. Selecting the best fluorescent protein for a specific application can seem like...under conditions that best mimic the intended application. The three key properties for live cell imaging...are converted into emitted photons. These are indicated by the extinction coefficient (EC) and quantum...experimental set up. The practical brightness takes all application specific parameters into account, including the... picture of what can be expected in a 'real' application than the theoretical brightness. Consequently...

...schematic depiction below): 1. Resistant cells among the separate replicates would contain the same labels...development of combination therapies.         References 1. Hyo-eun, C. Bhang, et al. "Studying clonal dynamics...summarized in a recent Nature Medicine paper. In the publication, Bhang and coworkers used the ClonTracer library...propagated it, separated it into roughly identical replicate groups, and treated these groups with the same...subpopulation 2. Resistant cells among the separate replicates would have different labels - i.e. they would... Looking at barcode abundances among multiple replicate populations, they found that similar barcodes ...populations were shared by at least one other replicate; prior to treatment, there are small (previously...

...Introduction to Biosensors. Essays Biochem. 2016 Jun 30;60(1):1-8. DOI: 10.1042/EBC20150001 Leopold, A. V., Shcherbakova...of the biosensors used in research are very sophisticated, but at their core they can all be boiled down...ready-to-use. While biosensors span the gamut of applications within chemistry, biology, and physics, in this...sensors can be targeted to different cellular locations or tissues depending on the biology you are studying...different factors, in different or the same cellular location, all at once! Small molecules What type of small...Neurotransmission and Neuromodulation: Engineering and Applications. Frontiers in Cellular Neuroscience. 2019 Oct...

...protein of interest will be problem-free. In figure 1 we provide an example of the OSER assay with a number...can be shared by publishing it as (part of) figure 1, uploading it as a preprint, or documenting it in ...aggregates. This property can be tolerated in some applications (e.g. labeling of cells or tracking promotor...promotor activity), but it is problematic in applications in which the fluorescent protein is used as an inert...During the experiment, the solution is diluted, complicating the interpretation. Because of this, these methods...aware that the fluorescent proteins reported in publications are only characterized under a limited number...build a valuable resource with tools and data that indicate the performance of (a set of) fluorescent proteins...

...to reach out to potential depositors in two ways: 1. Fill out our "Suggest a Plasmid" form Fill out the...suggestaplasmid, the name of the plasmid and a publication where we can find the plasmid. While we cannot...

...bioengineering, but it wasn’t clear whether they could 1) be decorated with proteins and remain functional,...Follow her on Twitter @jessicapolka.     References 1. Kanabrocki, J. A., Quackenbush, R. L. & Pond, F. ... modified to extend at different pH levels. Domesticating R bodies We have shown that all three of these...actuators that could be used in a variety of applications. For example, you could use them to lyse cells...matrices) in response to pH changes. Beyond these applications, purified R bodies could also perform as actuators...PubMed PMID: 26814170. Find Plasmids from This Publication at Addgene 4. Petroni, G., Spring, S., Schleifer...

... Zebrafish) (Xiong, et al., 2021).    Fig. 1: Schematic showing the workflow for the BLITZ system...Biol. 2022. https://doi.org/10.1038/s41556-021-00836-1 Visual barcodes for live cell clonal multiplexing...of interest and, for in vivo work, generate a dedicated transgenic line. Using zebrafish as a model organism...off-target mutations. These plasmids contain catalytically inactive dCas9 and microbial SSAPs (dCas9-SSAP...clonal multiplexing of cells in live imaging application. Each visual barcode consists of a fluorescent...transcription response element (TRE) or kinase translocation reporters (KTRs). Find visual barcode plasmids...

...non-functional allele. For a 1 kilobase gene, since potential target sites occur ~1 in every 8 nucleotides,...770–788 . https://doi.org/10.1038/s41576-018-0059-1 Veres A, Gosis BS, Ding Q, Collins R, Ragavendran ...the optimal location. It is also important to have good information on the exact location of the TSS. ...appreciable levels of modification. Whole-genome sequencing of cells modified by CRISPR indicates that the consequences...specific base edit, or modulation of gene expression. Location and sequence are important considerations for ... gRNAs. For indels, it's not so important what location in the gene you target, but it is important that...fewer sequences to choose from). Finally, for HDR, location is much more important because you have to target...

...Hsp70, the heat shock releases heat shock factor 1 (HSF-1), which subsequently binds to heat shock elements...prokaryotic promoter often used for bacterial protein purification. When arabinose is absent, regulatory protein...

...versions with toxicity to many organisms. Fig. 1: Exogenous sources of dsRNA Cellular sources of... 286-301 (2022). doi.org/10.1038/s41580-021-00430-1 More resources on the Addgene blog CRISPR 101: RNA...generate dsRNA as part of their infection and/or replication process within the host. dsRNA viruses are inherently...generate dsRNA intermediates as part of the viral replication process. dsDNA viruses yield dsRNA through a ...

...healthcare system was $35.2 billion (or $140 per $1 invested in the trial). The goal of gendered innovations...is the goal of Gendered Innovations. This project: 1) develops state-of-the-art methods of sex and gender.... To remedy this, Shah et al. provide a table indicating the sex of the most commonly used cell lines ... work to be done! Researchers need to learn sophisticated methods of sex and gender analysis. Universities...

... closely data points fall to each other (see Fig. 1). Alpha values – numerical risk allowance in a ...statistical test assumes your data follows. Fig. 1: Examples of comparable groups with and without equal...values from smallest to largest and ranking them (1 being the smallest value). It calculates the cut off...tests  Statistical tests fall into two general categories: parametric and non-parametric. In this blog ... exist somewhere within your data; it does not indicate which groups the differences exist between. For...that matter. However, this is oftentimes an oversimplification. If your independent variables are affecting...

... as a Professor. Step 1: Your PI forwards you a Request For Applications (RFA), what next? First,...proposal typically lists the deliverables, explains 1-2 potential pitfalls of the intended protocol, and...the grant application process. When my PhD advisor first asked me to write a grant application due the ...in a previously funded grant application to see how funds were allocated. Step 3: Get feedback from your...the next round of applications. Step 5: Keep writing! Each written grant application builds upon the ... project grant application" by the NIH Still want more information on the application process? What types...advisor alone applies for these competitive grant applications; however, in some cases, your advisor may choose...

...all the colors of the CRISPRainbow. Figure 1: CRISPRainbow was used to track the localization of...is available as a kit from Addgene.   References 1. Chen, Baohui, et al. “Dynamic Imaging of Genomic ... every genomic locus, and to do so in vivo. Catalytically dead Cas9 (dCas9) cannot induce gene editing...Cas9 ortholog, and the target sequence must be located adjacent to that PAM. To scale up fluorescent CRISPR...this rainbow of colors, Ma et al. performed sophisticated tracking of multiple chromosomal loci in live... do not induce genome editing. If used with catalytically active Cas9, the short, CRISPRainbow gRNA will... PMCID: PMC3918502. Find plasmids from this publication at Addgene. 2. Ma, Hanhui, et al. “Multicolor...

... the original plasmid (Option 1) or added using PCR-based amplification (Option 2). Advantages of Golden...Methods Mol Biol. 2014;1116:119-31. doi: 10.1007/978-1-62703-764-8_9. PubMed. More Resources at Addgene:  ... work around this is to "domesticate" your fragment: PCR-based amplification can be used to create silent...and exciting applications, this system is an incredibly powerful tool for cloning complicated constructs...Type IIS sites (such as BsaI or BbsI), that are located on the outside of the cleavage site. As a result...restriction sites that may not be amenable to "domestication", you might want to consider using an alternative...high-efficiency step. References: Sequential amplification of cloned DNA as tandem multimers using class-IIS...

... plasmid-based system for locating and containing escaped flies. Nature 4:1-22. https://doi.org/4.01.2022...Additional resources on the Addgene blog:  Flies 101: Catching flies in the lab References Abril, U.N.O., et....

...and accurately in a “single pot.’   Figure 1: Single-Pot Assembly: a) Graphic depiction of chromatin...AID system relies on a transport inhibitor response 1 (TIR1) expressed under a desired promoter with a specific...2021.   The potential tools fall into two categories: probes and editors. A probe array may consist...and should be varied as it can have important implications for the fusion protein effectivity), and a fluorescent...to visualize DNA, an editor targets DNA (via catalytically inactive dCas9) to modify chromatin. A fluorescent...both spatiotemporal protein degradation and quantification of this depletion using imaging-based measurements...BioTag, which can be used in protein affinity purification studies.    Duong et al., Genetics 2021. https...

...all of its successes, has many deep-rooted flaws (1). As we discovered through the four workshops at the... Follow David on Twitter @driglar.     References 1. Alberts, Bruce, et al. "Rescuing US biomedical research...University of Massachusetts Medical School. Graduate education programs are also beginning to address these issues...The panel discussion on Friday morning will be dedicated to Academic data and the Labor Market. On this...Sinche is a STEM career counselor, Director of Education for the Jackson Laboratory for Genomic Medicine...disease.  Outside the lab, he is interested in communicating science and in the intersection between science...

...screening blog post for more information.   Figure 1: Result of a blue-white screen. Image from Stefan ...Because Sanger sequencing can usually sequence just 1 kb of DNA, custom primers are especially helpful in... of the entire plasmid. Please note that NGS verification is more time-intensive than Sanger sequencing...

...additional flexibility in pseudotyping.   Figure 1: Genome of a prototypical gamma-retrovirus.    ...an SV40 promoter, and Neomycin selection.   Table 1: Gamma-retrovirus glossary Plasmid Type Element... be used interchangeably. Are γ-retroviruses replication incompetent? Yes, as with lentiviruses, the genes...

...Last updated Sept 1, 2020. This post was contributed by David Wyatt and Dale Ramsden, UNC at Chapel Hill...the course of repair by NHEJ are typically small (1-10 bp) but extremely heterogeneous. There is consequently...Giovannangeli C, Jasin M, Brunet E (2014) Chromosomal Translocations in Human Cells Are Generated by Canonical Nonhomologous...

...are working with is correct. It's also best to use 1-2 ug of the vector for digestion. PCR Design primers...plasmid; a starting point could be a molar ratio of 1:2 (plasmid:insert), but this parameter has to be optimized...constructs. His work was acknowledged in hundreds of publications including Cell, Nature, and other journals. ...

...introduced, they can deplete endogenous protein levels for 1-3 days. There’s also a CRISPR for that! CRISPR interference...more about them on our CRISPRa blog! Fig. 1 CRISPR activator and repressor mechanisms Want...interference (CRISPRi) targets a catalytically dead Cas9 fused to a transcriptional repressor to your ...

...main differences between Cas7-11 and Cas13 are 1). non-specific RNA cleavage and 2). cell toxicity. Cas7... targeting single stranded RNA.   Table 1: Fast facts on Cas family proteins and genome editing...a defined site to form a clean DNA break, Cas3 locates its target sequence and initiates single stranded... out large regions of DNA and has anti-viral application potential as a result. Fast facts – PAM requirement... has been proposed to be useful for the same applications (RNA knockdown, RNA editing, etc.). The main...

...the lab running like a well-oiled machine. Lab tip #1: Glove trick Putting on lab gloves can sometimes be...With barcode tracking, you will always be able to locate materials quickly and easily. Learn more about ...Additional Resources on the Addgene Blog New Educational Resource: The Addgene Videos Page 5 Science Rap...

...company culture.  It’s also a good idea to prepare 1-2 unique questions for each interviewer by looking...to take notes after each interview.   Tailor your 1-hour presentation to your audience Start by asking...accomplishments and demonstrate your science communication skills. Know your audience. Connect your prior...codes for interviews. You can also ask HR for clarification on the dress code for the interview. Greet each...

...writing and hopefully increase your success rate. 1. Submit your grant early! First off, and perhaps the...involved so it’s important not to throw in the towel if 1 or 2 funding bodies say no. If possible, get feedback...Series 4. Become a science communicator More and more fellowship applications are asking for a 200-300 ...good quality application. Even following this advice, only about 50% of my grant applications have been ...do need to be able to research and write your application, then give it to someone else to read and / or...it). It’s easy for reviewers to spot when an application is rushed (whether the idea isn’t properly researched...researched / developed or the application is full of typos) and these are unlikely to get past the initial...

...self-assembly for applications in health, energy, and sustainability.   References 1. Pardee, Keith, et...detects samples at clinically relevant concentrations (1-3 fM; 1x106-fold lower than before), and our combined... journal Cell just six weeks later. The Zika publication timeline I think we can speak for all the authors...research, but the urgent need for the test did catalyze our resolve to get the work done as quickly as... dozen versions of the toehold switches and amplification primers. This helped make sure that we had a...this includes primer directed isothermal RNA amplification, new toehold switch designs, CRISPR/Cas9-mediated...our electronic reader that provides portable quantification of diagnostic results.  Our approach now detects...

...: it’s dCas9… with no modifications). Ready to get started?   Figure 1: Break free from the ball...?  Dividing cells depend on DNA methyltransferase 1 (DNMT1) to copy methylation marks from parental to... when DNMT1 methylates the genome during DNA replication, it doesn’t methylate sites blocked by dCas9....for dCas9 to interfere with DNMT1 even through replication. The dCas9 also needs to be removeable, otherwise...vivo using dCas9–peptide repeat and scFv–TET1 catalytic domain fusions. Nat Biotechnol 34, 1060–1065. ...

...Emerging Investigators Podcast breakdown Intro Chapter 1 - Introduction to Mark Springel, Olivia Ho-Shing, ...Additional Resources from the Addgene Blog Find education resources Use our Plasmids 101 eBook to teach ...

...Fragment? Consider the classic IgG antibody (Figure 1): it is composed of four peptide chains (two identical...that actually bind antigens.      Figure 1: IgG antibody structure with important features labeled...fragments described above can be used for similar applications and have similar advantages, though with some...

...Glycine-HCl 25–200 mM SDS 0.1%–1% (w/v) Tween 20 (optional) 1% (v/v) HCl Adjust to pH 2.0... tool to identify a protein of interest in a complicated solution like cell lysate. But they can be a ...

... Joachim Goedhart Podcast breakdown Intro Chapter 1 - How Joachim ended up at the University of Amsterdam...Twitter and has helped us update many of our educational resources. Listen to hear all about Professor...speeds science. Chapter 8 - The importance of communicating research findings through social media and other...

...forms of science communication. Click here to listen to the interview with Tom McFadden!   1. Rosalind Franklin... from Addgene.      Check out more science education tools like Verse Two and Fill in the Planck at...with us. Tom McFadden is a YouTuber and science educator looking for creative ways to implement the next...next generation science standards and communicate science. Learn more about Tom on his youtube channel ...Community Check out All of Our Podcasts Find Great Educational Content in our Plasmids 101 Series Additional...

...phosphorylated DNA termini to 3 ́-OH DNA termini (1-4). In vitro, ligases (notably T4 DNA ligase) are ... enzymology of DNA repair enzymes.     References 1. Shuman, Stewart, and Christopher D. Lima. "The polynucleotide...thermostable DNA ligase-encoding gene." Gene 109.1 (1991): 1-11. PubMed PMID: 1756968. 7. Lauer, G., et al. "Cloning...or DNA base pair mismatches (8-11). For some applications, there cannot be any end-joining activity, and...regions. Why use a high fidelity ligase? Numerous applications have been developed to take advantage of the...Taq and other thermostable DNA ligases. These applications detect specific nucleotide sequences with high...rounds of melting and annealing allows linear amplification of the probe ligation product. With the right...

...CRISPR tools.  Distributing and depositing In Figure 1, you can see cumulative numbers of CRISPR plasmid ...new CRISPR depositors each year.    Figure 1: Addgene CRISPR distribution and deposits. (A) Cumulative...estimated scatterplot smoothing. (C) Number of requests per year for pooled libraries, categorized by function...insertion via a chemical modifier. We used these categories to see what kinds of CRISPR tools people were... pooled libraries, although we used different categories (Figure 2C). Note that in Figure 2, the numbers...Cumulative number of deposited CRISPR items, categorized by CRISPR function, by year. (B) Number of requests... years.  Libraries are divided into different categories based on function: knockout (KO), activate, inhibit...

...interrogated by the optical system (Figure 1).   Figure 1: Cells expressing a particular surface ...populations of cells in a complex tissue or sample (1). When combined with cell sorters, this technology... it passes through and scatters in different directions. The degree of scatter is captured by detectors...Light that scatters sideways to the path that it was initially traveling is called side scatter (SSC) and...granulocytes in a plot of forward scatter (FSC) versus side scatter (SSC). Users can gate around their...protein composition. It is used for a variety of applications such as measuring target protein expression ...expression levels, assessing post-translational modifications, determining cell health, analyzing cell cycle stages...

...principles of fluorescent dyes/ligands Figure 1: Example structures of fluorophores, fluorescent dyes... dopamine D2/D3 receptors. Scientific Reports, 10(1). https://doi.org/10.1038/s41598-020-78827-9 Breen... Biophysica Acta - Biomembranes (Vol. 1838, Issue 1 PARTA, pp. 15–33). https://doi.org/10.1016/j.bbamem...emission wavelengths, which are pivotal in their application in biotechnology (Grimm & Lavis, 2022). These... substrates/peptides and antibodies. Example applications of this include coupling to an antibody, whereby...whereby excitation of blue light will indicate where FITC is bound through green light emission. Key considerations...typically bind to. Stability of fluorescence: For applications that involve ranges of pH, and significant lengths...

...found climbing mountains or playing with his two cats. Keira Havens grew up in Hawaii where she was fascinated...challenges involved in plant synthetic biology. Challenge #1: Public perception of synthetic biology One of the...biotechnologists face is public perception of genetic modification (GM) and synthetic biology. The mistrust and...cannot build what we do not understand, and the complicated and unique metabolic processes of plants are ...

...editors (ABE) mediate A•T-to-G•C base changes (Figure 1), but it can be challenging to make these base changes...dCas to the genomic DNA of interest  Figure 1. When ABEs are in action they bind DNA so that the... from E.coli, named TadA7.10 in ABE7.10)  a catalytically impaired Cas protein (dCas or Cas nickase)  ...accidental insertions and deletions (indel) by the catalytically impaired D10A nickase from ABE7.10 can be a ...the authors created ABE8 constructs using a catalytically “dead” S. pyogenes Cas9 (dC9-ABE8.x-m/d). The...these variants, ABE8 could target many additional locations in the genome. Compared to ABE7.10, NG-ABE8.xm... reduced off-target editing frequencies. For applications that require high DNA editing specificity, for...

...fluorophores are constantly being engineered. Table 1 lists a few common fluorescent proteins and their ...definitions found in Table 2 may not apply. Table 1: The specific mutations comprising common fluorophores...transgenic mice. Purification: GFP can be used as a general epitope tag for protein purification and a number...their experimental needs, and some of the common applications in the lab. Why Green Fluorescent Protein?...to maintain fluorescent activity; very little truncation is tolerated, however, point mutations are acceptable...Re-engineering GFP to increase its range of color and application Almost as soon as its sequence was elucidated...others) Neutral mutations    A multitude of applications Due to its size and ease of use, GFP and other...

...cleavage (Harrington et al., 2017). Figure 1: Anti-CRISPR proteins (Acr's) can block CRISPR activity... ways to anti-CRISPR proteins in your experiments 1. Reduce off-target effects Prolonged Cas activity ..., 2018). For example, AcrIIA4 mentioned above indicates that it’s the fourth Acr protein discovered that...viral particles that have the gene deletion could replicate when challenged with the S. islandicus CRISPR-...B, Bondy-Denomy J (2020) Anti-CRISPR protein applications: natural brakes for CRISPR-Cas technologies....light-inducible Cas9 activity? Check out these these plasmids Catch Up on Your CRISPR Background with Our Guide Pages...

...crevices proteins on the cell surface. Figure 1: Comparison of common antibody, HCab, and nanobody...Humana Press, pp 133–148. https://doi.org/10.1007/978-1-62703-293-3_10 Walter J et al. (2020) Synthetic nanobodies...Starting with libraries and a biotinylated (for purification purposes) protein target of interest, the protocol...-30) of sybody candidates are sequenced for identification. All unique sybodies can be subcloned into ...2020) Generation of synthetic nanobodies against delicate proteins. Nature Protocols 15:1707–1741. https...Light to Manipulate Cell Signaling and Protein Purification RANbodies: Reporter Nanobody Fusions Plasmids...

...Updated Jun 1, 2021 by Meghan Rego. Reproducible data are key to science, so scientists are used to repeating...the DNA packaged inside the viral particles (Figure 1). Briefly, packaged DNA is extracted from purified...These impurities cannot be removed during the purification step because they are inside the virus itself...very low concentration. So how do we make sure to catch all of these potential mishaps before distributing...Guerin et al., 2020).  In addition to genome identification, we developed custom open-access Python scripts...

...used in way more applications.   Fig. 1 Making a Protein Tag (Abridged) (1) Locate a promising monoclonal... by its antibody without additional chemical modification steps and when the epitope was within the tagged...

...Podcast breakdown Intro Chapter 1: Who is Crystal Shih? Crystal's educational background and position at Novartis...Chapter 7: How Crystal developed her science communication skills and how they've helped her at Novartis...

...for example, split cells on a Monday diluting them 1:10, you should be able to split on the following Thursday...Immunology. You can find Sana on LinkedIn.   References 1. Yin, R., et al. "Gene expression profiling analysis...results. Most cell culture media contain the pH indicator phenol red (pH range 6.0 (yellow) - 8.0 (red))...Anything between orange and light orange usually indicates that the cells have grown enough, are making their...

...DYKDDDDK or DYKDDDK 1 Affinity and Purification Good for antibody-based purification; has inherent enterokinase...does not greatly alter the size of the tag.   Table 1: Common protein tags Tag Epitope Mass (kDa) Function...site GST Large Protein 26 Purification and Stability Good for purification with glutathione; protects...used for purification as elution requires low pH poly His HHHHHH  0.8 Affinity and Purification Very small...GKPIPNPLLGLDST   1.4 Affinity and Purification Good for antibody-based purification Combo and cleavage tags...multitude of uses including (but not limited to) purification, detection, solubilization, localization, or...fusion proteins to aid in the expression and purification of recombinant proteins. Tags for stability...