...Waugh, 2002). The tag can also be proteolytically cleaved after purification, so as not to perturb protein...for downstream applications. Tag cleavage must be performed after primary purification and requires a ...thoughtful planning, affinity tags can make protein purification a cinch. Types of affinity tags, the proteins... Affinity tags are used primarily as protein purification tools (and can have secondary functions as detection...single protein is expressed, with the intent of purification, it will be expressed along with all other cellular...available antibodies, resins, beads, and other purification aids readily available. Choosing the best tag...tag to remain on your protein for downstream applications Below, we describe a few of the most common...

...the sandwich and typically takes less than an hour. However large proteins typically do not transfer as...validated applications, pay close attention to the controls listed on the vendor’s website. Typically, the ...downregulated in a system, detect post-translational modifications, quantify protein levels relative to standards... boiling in the presence of a reducing agent, typically containing thiols, to cleave disulfide bonds. ...gel on top of the resolving gel. A stacking gel typically has a different ionic strength and lower pH and...are “sticky” and have the potential to bind nonspecifically to the antibodies used for staining. To address...search, first pay attention to the validated applications listed on the vendor’s website and choose an...

...would be a good time to talk about the amplification and verification processes we use to ensure high-quality...your own experiments. Library amplification 101 Library amplification seems straightforward: E. coli ...represented. Typically, Addgene uses electrocompetent Stbl4 cells when performing amplifications, and we use...transformed with the library DNA, they grow and replicate, and then their DNA is harvested, much like one...library has its own depositor transformation and amplification protocol, but the overall workflow is similar...ll need to remember when undertaking library amplification and sequencing. Maintain representation of the...ensure a good transformation efficiency. For a typical library, Addgene usually does four to eight electroporation...

... expression for experimental, biotechnological, and medical applications. Researches can visualize proteins...of complex post-translational modifications. Extraction and purification of recombinant proteins from ...of proteins including antibodies and pharmaceuticals, specifically interleukins. Yeast expression systems...downstream applications. Cell-free expression systems however, are expensive and very technically challenging...modified via an array of post-translational modifications including protein folding, formation of disulfide...mRNA and the addition of post-translational modifications Learn more molecular biology in our Plasmids...expression systems used for protein production and purification. These include mammalian, insect, bacterial,...

...or macromolecule. Biotin can be added enzymatically or chemically. Enzymatic biotinylation occurs naturally...molecular biology applications for biotin and avidin. A short list of some other common applications and links...downstream application. Biotin labelled oligonucleotides can be used in a multitude of applications including...post-translational modification of exceptional specificity.” Trends in Biochemical Sciences, 24.9 (1999b... M. “Biotin: The forgotten vitamin.” American Journal of Clinical Nutrition 75.2 (2002): 179–180. PubMed...dye-labeled molecules on surfaces.” Journal of the American Chemical Society 125.27 (2003): 8074–8075. PubMed PMID...this highly specific post-translational protein modification. Most organisms have less than five enzymes ...

...colleagues have developed a new genetically-encoded voltage indicator (GEVI) called JEDI-2P (Liu et al...Verkhusha’s lab has generated a new genetically encoded calcium indicator using small monomeric NIR fluorescent...Tuning the Sensitivity of Genetically Encoded Fluorescent Potassium Indicators through Structure-Guided...colleagues present an improved genetically encoded temperature indicator B-gTEMP designed to meet those... B-gTEMP itself is a significant improvement over previous temperature indicators, it also demonstrates...doi: 10.1038/s41592-022-01597-x. New glutamate indicator iGluSnFR3 Improving on the glutamate sensor iGluSnFR...They generated variants optimized for different applications (v82 has higher affinity for glutamate but slower...

...process allows you to physically magnify a sample so you don’t have to optically magnify quite so much...compatible with conventional antibodies, chemical dyes, and genetically encoded fluorescent proteins (Gambarotto... and the physical properties of the sample itself. For example, cultured cells are typically easier to...promises an easier, more accessible way to image biological features previously only visible via techniques...with an abundance of protocols and examples of applications from across disciplines now available. In this...experiment or evaluating ExM images in a new publication.         Why Expansion Microscopy?  Traditional...techniques also require specialized instruments and technical expertise, limiting their accessibility (Prakash...

... and LV have been used successfully in preclinical and clinical settings for ocular conditions. However...used in clinical trials, a variety of engineered ones have shown great promise in preclinical studies ...that I got into the field of gene therapy, more specifically AAV-based retinal gene therapy. The year was... Gelsinger in an adenovirus-based gene therapy clinical trial gone horribly wrong. It brought the whole...scientists, doctors and patients hope are many more clinical successes for gene (and cell) therapies. One may... over 25 years ago when completing her PhD and medical training in Ophthalmology. Gene therapy was in ... time advances in human genetics led to the identification of several disease-causing genes, including...

...dCas9-fusion proteins has dramatically increased the potential applications of CRISPR/Cas9 in genome-engineering... tools for all-optical electrophysiology A major challenge in the field of all-optical electrophysiology... the perfect voltage indicator and optogenetic actuator pair. Ideally, indicators should possess fast ...sensitivity. This hierarchical screen led to the generation of two enhanced voltage indicators: QuasAr1 and...temporal control over CRISPR/Cas9 mediated genome modification and gene expression. The plasmids associated... system widely accessible for many potential applications. Polstein LR & Gersbach CA, Nat Chem Biol ...expression Brilliant technologies adopt basic biological concepts and employ them in creative and innovative...

... editing in Escherichia coli. Acta Biochimica et Biophysica Sinica. https://doi.org/10.1093/abbs/gmab036...far, we’ve used the MUCICAT system in two ways: In our initial paper, we used MUCICAT to optimize the gene...industrial strain. Not only is MUCICAT better, it is faster. Using MUCICAT only needs 2 newly constructed...engineered cells for green chemistry and biomedical applications.   Yiwen Zhang entered the Sheng Yang Lab...integration using CRISPR-associated transposases” or MUCICAT. Here, we’ll describe each of these CRISPR systems...providing users with a complete editing process. The MUCICAT system While the above systems are great for experiments...chromosomal integration (ex: genome editing), our MUCICAT system which is based on the CRISPR-associated ...

...SR, Bird A (2017) Radically truncated MeCP2 rescues Rett syndrome-like neurological defects. Nature 550...awareness and published his observations in a German medical journal. Decades later and after years of painstaking...silenced by a Lox-Stop cassette, resulting in the neurological deficits observed in Rett Syndrome. Restoring... tamoxifen-inducible Cre not only halted the neurological damage but completely reversed it, regardless...this interaction to varying degrees determine the clinical severity of different MECP2 mutations. More recently...the MECP2 protein is necessary to rescue the neurological defects of Rett syndrome (Tillotson et al., ...detrimental as too little, resulting in MECP2 duplication syndrome or Rett Syndrome, respectively. One ...

...regulator. Other methods for physical shearing exist as well, such as sonication and needle shearing. Essentially... whole genome amplification (by a mechanism known as multiple displacement amplification) even in the ...proteins! Many thanks to our guest blogger Jessica Sacher! Jessica Sacher is a microbiology PhD student at...Their Viruses." Chemical Reviews (2016). PubMed PMID: 27319741. 3. Simpson, David J., Jessica C. Sacher, and...This post was contributed by guest blogger, Jessica Sacher, a microbiology PhD student at the University...bacteria) may be the most numerous and most diverse biological entities on our planet, but we still know collectively...adding methyl groups, glycosyl groups, and other modifications to their bases in order to protect their DNA...

...them. An easy way to do this is gel purification. In gel purification, you use a voltage difference across...backbone. Sample results indicative of successful and unsuccessful ligations are indicated below. A successful...grow overnight cultures for DNA purification. The simplest purification you can do is a miniprep, but if... maxiprep. In these purifications, you generally lyse the bacteria; add chemicals to precipitate out the...that can be easily maintained in a variety of biological systems. Read on for an in-depth breakdown of...digests. Of course there’s much more detail and verification required for the process to work well, so let...backbone. Because you lose some DNA during the gel purification step, it is important to digest plenty of starting...

... microscopy technique to incorporate optical sectioning. Optical sectioning refers to the ability to extract...Super-resolution microscopy uses a number of optical and chemical ‘tricks’ to turn subsets of fluorescent ...Richardson, Director of the Harvard Center for Biological Imaging and a Lecturer on Molecular and Cellular...are a sports photographer at the Super Bowl, a medical technologist taking an x-ray, or a biologist imaging...excitation volumes to enhance spatial contrast (i.e. optical sectioning (2) or super-resolution (3). Fluorescence...before reaching the detector. The pinhole is specifically placed to only allow light from the focal plane...of a sample. Therefore, when a large number of optical slices are acquired all fluorophores throughout...

... sport athletes, as well as the ethical and health policy implications arising from sport-related concussion... years of exclusively writing lab reports and empirical essays had also stirred some of my more creative...begun my journey towards a career in science communication, an established field of inquiry with which ...University of Otago’s Department of Science Communication the following year and, having thoroughly enjoyed...straight on to a master's degree in science communication. The first year of the course involved a whirlwind...world of industry. Along this road, science communication graduates are often well-equipped. Many institutions...truly benefit others. Herein, a good science communicator—with an understanding of how to appeal to a ...

...transmitted transsynaptically). This is because the recombinant forms of these viruses typically used in ...able to replicate, and for a virus to cross a synapse from one cell to another, it must replicate. Modified...tracing, it has two practical limitations. First, these high levels of viral replication are toxic. Second... well as to only spread monosynaptically from these cells. These modifications take advantage of the fact... became foundational later on when we studied biological processes, like how our stem cells are housed... thus, are a major part of the answer to many biological questions about the brain. To address this, molecular...enter cells and deliver a variety of cargoes, genetically engineered neurotropic viruses emerged as powerful...

...desired modification. Finally, the positive clones are sequenced to confirm the desired modification and ...polymerase activity (for amplification), 3’->5’ exonuclease activity (increases amplification fidelity), and no... genome modifications. Occasionally, multimerization of the PCR primers can cause duplication of the primer...the desired modification have been identified (and confirmed to be free of primer duplications), final validation...used to mutate the PAM sequence (an NGG sequence critical for Cas9 cleavage), thereby rendering the resulting...and the absense of additional modifications.   Site-directed mutagenesis of Plasmids. A) Generation..., which flank the targeted region. Upon PCR amplification, the template is removed by DpnI restriction...

...recombination. Yeast Replicating plasmids (YRp):  These vectors contain an Autonomously Replicating Sequence (ARS...These vectors replicate as though they are small independent chromosomes and are thus typically found as a...not necessarily promote replication.  Selection markers for yeast Historically, scientists have utilized...mammalian vectors, will catch you up on the core replication and resistance features of yeast vectors and ...proteins yet they still share many of the same technical advantages that come with working with prokaryotes...includes but is not limited to: rapid growth, ease of replica plating and mutant isolation, a well-defined genetic...mutation. Yeast expression plasmids used in the lab typically contain all the necessary components to allow ...

...a tail, and when gap junction-mediated electrical communication is briefly inhibited in the entire worm...fluorescent reporting and genetically encoded fluorescent voltage indicators (GEVI) are ideal tools to...thing that comes to mind. However, ion-based communication is not just for neurons. Non-neuronal cells ...It's just that for non-neural cells, ion-based communication happens at a slightly slower scale.  All living...living cells with a membrane possess an electrical state. This state, known as the resting potential (... (Vmem), can be measured as the imbalance of electrical charge between the inside and the outside of the...bioelectricity is revealing the important role of bioelectrical signaling mediated by ion-channels and pumps...

...systems have been used as biological tools for a wide range of applications in molecular biology or in...activity of CcdB. Applications of ccdB in plasmid cloning Perhaps the most widely used application of CcdB is...selection markers. Restriction modification systems Because restriction modification systems consist of two enzymes...types of plasmid addiction systems and their applications. The discovery of toxin-antitoxin systems Toxin-antitoxin...CcdA (antitoxin) where CcdB inferes with DNA replication by binding the GyrA subunit of DNA gyrase and...construct. Other toxin-antitoxin systems and their applications MazE/MazF Aside from CcdB/CcdA, there are many...counterselectable marker to create unmarked genetic modifications in the yeast Pichia pastoris. By combining both...

...are used across species: replication, selection, and expression. Replication It’s usually not enough to...chromosome has a single origin of replication (ori), where DNA replication begins, so a bacterial plasmid...Eukaryotes are a bit more complicated. Yeast vectors may contain an autonomously replicating sequence (ARS), a...fast-growing populations of cells, the vector needs to replicate alongside its host, or it will be lost from the...compatible ori that will be recognized by its host’s replication machinery. A shuttle vector designed for unrelated...experiment. Yeast shuttle vectors have these replication sequences for their yeast targets as well as ...episomal vectors is to include viral oris to drive amplification, which helps increase cellular uptake, but this...

...Each round of amplification should result in a 10-100-fold increase in virus. Purification (2 days) - Purification... Purification is required if rAdV is to be used in vivo. The standard method for purification of rAdV ...or 10 days) Physical titer (particle count, rAdV genomes/mL) can be measured by: Optical density: particle...vectors (AdV) are attractive vectors for research applications and gene therapy: they can be produced at high...AdVs is what makes them ideal candidates for applications in oncolysis and vaccination!) In the 65 years...years since its discovery, researchers have done significant work to improve the Adv genome’s utility in research...Adenovirus-mediated gene delivery: Potential applications for gene and cell-based therapies in the new...

...are currently approved or have made significant progress in clinical trials.     Figure 1: Types...editor-based clinical trial for SCD in 2022 and Editas Medicine starting a Cas12-based clinical trial for...choice. In sum, CRISPR therapies have made significant clinical strides since we first wrote this blog post... no longer an accurate descriptor; progress in clinical therapy is more fitting, so it is time to update...some solid tumors are currently in early-phase clinical trials and have shown promising preliminary results...introduction of the cancer-fighting component, CAR. Clinical trials are underway to explore CRISPR’s capacity...for these diseases was sponsored by Vertex Pharmaceuticals and CRISPR Therapeutics. Data shared from these...

...Nature chemical biology. 2021 May;17(5):509-18. Inoue M. Genetically encoded calcium indicators to probe...Iino M. Genetically encoded fluorescent indicators for organellar calcium imaging. Biophysical journal...sensors. Selecting a calcium sensor Genetically encoded calcium indicators (GECIs) are fluorescent proteins...transients in living cells and organisms. Unlike chemical indicators, these DNA constructs can be delivered to... Imaging neuronal activity with genetically encoded calcium indicators. Cold Spring Harbor Protocols. ...Measuring calcium signaling using genetically targetable fluorescent indicators. Nature protocols. 2006 Aug;...studies, you can start by asking ‘What is the biological phenomena I want to observe?’ This information...

... capabilities. Specifically this includes primer directed isothermal RNA amplification, new toehold switch...portable quantification of diagnostic results.  Our approach now detects samples at clinically relevant...global strains of the Zika virus (Asian-, African- and American-lineages). Strain discrimination is especially...current outbreak because of the neurological symptoms associated with the American-lineage of the virus. For...populated regions of South, Central, and North America. Diagnostics will play an important role in helping...outbreak was reported, and we felt that, given the critical need for low-cost, field-based diagnostics, our... journal Cell just six weeks later. The Zika publication timeline I think we can speak for all the authors...

...methods of replication. In fact, an entire classification scheme, known as the Baltimore replication classes...that in a bit! Viral genomes and replication When it comes to replication, viruses tend to march to the ...developed around how they replicate (Figure 2). Figure 2: Baltimore replication classes. Image credit: ...an intermediary DNA replication step between DNA and mRNA. DNA genome replication can involve an RNA or...that have been genetically modified to either limit or completely eliminate their replicative ability. The...Viral Vectors 101: Viral Applications Viral Vectors 101: Viruses as Biological Tools Viral Vectors 101:...cell. The cell then uses its own machinery to replicate the viral genome and proteins. This is, for the...

...understanding of complex biological processes. Many tools exist to perturb biological function in a direct...use in dsRNA applications. Figure 1: Schematic of an RNAi-inducing transgene. Typically, the desired...individually engineered and empirically evaluated for their gene silencing efficacy, thereby requiring a substantial...limitations of any technique in the context of the biological question. For example, while gene knockouts have...makes traditional knockouts unattractive from a practical perspective. Therefore, techniques that function...degradation, blocking translation, or chromatin modifications of complementary gene sequences (Figure 1). ...silencing can be easily induced by exogenous application of long or short dsRNA. However, it is important...

.... The overlap here is that it is unethical to use scientifically unproven technologies to edit a human...human clinical trials. This model also demonstrates that when we are at the point of scientifically rigorous...throughout the history of medical research, there have always been people who unethically experiment on humans...Cas9 genome editing is not far enough along, scientifically, to be doing in humans; We have not had a broad... recently published a report on the social and ethical issues of genome editing and human reproduction...with healthy mitochondria, with the intent to eradicate mitochondrial diseases. The key success of the...investigate the scientific possibility, safety, and efficacy of the mtDNA transfer options being investigated...

...cell lines (European, East Asian, Indigenous American, Africa, or South Asian) used in the screens based...Sean and Aaron knew that their study had significant statistical limitations, and expected to find only ...kind of biological framework, suggested to them that their findings may be the result of a technical artifact...Biomedical research has a long and well-documented history of racial bias, which still impacts and influences...Dependency Map cancer cell lines (left). Heatmap indicating the breakdown of ancestry associated dependencies...Seeking an explanation  First, they looked for a biological explanation, a relationship between the genetic...machinery where to cut.      Fig. 2: Heatmap indicating loci where SNPs reside on sgRNA target sequences...

...next logical step after technical mastery. However, being a great scientist doesn’t automatically translate...cohesive team environment. Neglecting communication skillsClear communication is the backbone of effective management...transition While a PhD equips you with exceptional technical skills, transitioning into management often means...stepping into unfamiliar territory. Unlike solving technical problems in the lab, management involves a different...time, I discovered that I not only enjoyed the technical aspects of my work but also found fulfilment in...your team's—can lead to frustration. Clinging to technical tasksDelegation can feel unnatural for those accustomed...myself to focus on my core responsibilities, which typically revolved around the broader vision rather than...

...weather befitting of the ominous political climate in which American scientists find themselves today,...we spend years being taught to communicate in a very specific, technical manner, so much so that we often...also a science communicator, UBC Public Scholar, and a member of the Vancouver SciCATs. Follow her on ...March for Science from researchers all over North America. I hope that these perspectives, the optimism they...Alina Chan is a postdoctoral researcher at Harvard Medical School working on human artificial chromosomes....election.   Even as we marched in solidarity with our American neighbours, the fight is far from over here at...April because devaluing science has alarming implications for our public policies—many of which impact...

...your specific application. Consider also that there may be variations within an application. Take immunohistochemistry...website highlights a variation of your application, a publication successfully used it in a similar way...exciting new project aimed at understanding how a critical cellular pathway is regulated. You’ve read all...choosing an appropriate antibody first look at the application-specific data provided on the manufacturer’s ... a linear antigen tend to work very well for applications that denature a protein to its primary (linear...the most similar to yours. Take a look at the publications cited on the manufacturer’s website, which may...plan to. To secondary or not to secondary?  The application will also determine whether you should use an...

...regular part of basic biological research as well as clinical therapy. These biological tools are useful because...being engineered as biological tools, thought was given on how to keep the replication of virus and its ...the component that physically integrates into the host genome, the virus is replication incompetent, meaning...envelope plasmid), and the polymerase (Pol) for replication and Gag structural protein on yet a third plasmid...meaning it can’t replicate and continue the infection cycle once inside the host. While the virus can still... this safety feature prevents the virus from replicating and spreading in non-target cells, including ....    Viral cargo-specific safety   After replication competency, the second concern with virus safety...

...Finding an antibody that works for your application, specifically detects your protein, is species compatible...(stay tuned for our affinity purification tag blog!).   Applications of epitope tags   The...protein purification is not ideal with Myc, as low pH conditions are required during the purification, which...and have been widely used across species and applications. In this blog, we will review the most common...that are bound by antibodies for the purpose of purification or detection of the tagged protein. These peptides...not exist, or they don’t exist for a specific application, or they exist but are unreliable for a variety...almost certainly already been optimized for your application. Depending on the tag, directly imaging or tracking...

...of its biggest limitations is that sequence modifications can only be made at restriction enzyme cut sites...homologous recombination. It allows for direct modification of DNA within E. coli and is independent of ...engineering.  It can be used to make an assortment of modifications: insertion and deletion of selectable and non-selectable...however, only Beta is required when generating a modification with an ssDNA substrate.  Gam: Gam prevents ...insert site (Figure 2, top).  These primers are typically ~70 nucleotides long (20 nucleotides that anneal...regions flanking the target site).  The best applications for dsDNA inserts include: large insertions ...fragments, such as gene replacements and tags.  The typical frequency of recombinants is 1 positive clone out...

...2012) scFv Antibody: Principles and Clinical Application. Clinical and Developmental Immunology 2012:1...therapeutic applications where longer retention times often increase therapeutic efficacy (Ahmed et al...scFvs are often genetically fused to other proteins, which has led to their application for basic and translational...cheaper to generate since it’s genetically encoded.   Translational applications of scFvs: Just like antibodies...blood clearance, which is useful for imaging applications; better tissue penetration which is useful for... for therapeutic and imaging applications; and reduced immunogenicity when administered in vivo due to... translational research.  Basic research applications of scFvs: The SunTag system uses an scFv to amplify...

.... Lajoie, Marc J., et al. "Genomically recoded organisms expand biological functions." Science 342.6156...most inclusive databases of post-translational modifications, identifies a staggering ~250,000 phosphorylation...phosphorylation events on the activity of a given protein? Classical methods for studying protein phosphorylation A...steric and charge-based nature of the phosphoryl-modification; they are both much smaller, and carry only ...thus cannot reflect the true state of protein modification within the context of its appropriate regulation...plasmid. This includes 4 copies of a tRNA that specifically recognizes amber stop codons (TGA) to encode...effects on cell growth. The C321.ΔA strain was genomically recoded to eliminate all 321 amber stop codons...

...ASAP5, a faster and more responsive genetically-encoded voltage indicator (GEVI) than previous versions. ASAP5...responsivity in screening ASAP3 variants. B) Electrical and optical recordings of spontaneous activity in human...protein-coupled receptors (GPCRs) are critical components of many physiological pathways and represent the largest...by endogenous GPCR. Example applications include large-scale pharmacological profiling or interrogation...single-molecule biophysics Fast and responsive voltage indicator ONE-GO biosensors kit Antibodies for neurodegeneration... an enhanced pU6cm promoter, viral replicon amplification of cargo, and heat treatment.  Figure...Arabidopsis, suggesting the improvements may be broadly applicable to dicot species. In addition, they generated...

...Foundation, an organization working to advance biological engineering openly so as to benefit all people...Initiative. Endy's team made news in March with a publication in Science describing the development of “transcriptors...with the BioBricks Foundation to distribute the physical DNA samples, for when that's the preferred mode...parts must flow.' People must be able to use genetically encoded functions - the words in our language...operating system for engineering an entire cell. Typically when people use plasmids, they are trying to do...the entirety of a cell. That's a big long-term technical challenge. We have a strategy that organizes how...work, and it begins with the central dogma - replication of DNA, transcription of DNA to make RNA, translation...

... ITRs serve as the origin of replication and are required for replication, packaging, and vector persistence...viral rep (replication) and cap (capsid) proteins, which bind to the ITRs and initiate replication. ITRs are...important elements of AAV and rAAV vectors, modifications can drastically affect gene transfer efficiency and ...Adeno-associated viruses (AAV) are an important biological tool that allow scientists to express a gene ... with biorender.com. ITR structure  ITRs are symmetrical 145-nucleotide sequences that flank the ends ...binding site for Rep78 and Rep68 to initiate replication and a terminal resolution site that serves as...are noted. Created with biorender.com. ITR modifications and types  Since ITRs are one of the most important...