... systems that measure biological phenomena by generating an output which is directly proportionate to ...antibody, enzyme, DNA, etc. The receptors job is to generate a quantitative signal upon recognizing the analyte... (fluorescence, pH, etc.). The transducer will generate a numerical value which represents the amount ...disciplines! Most of the sensors designed for calcium generate a fluorescent output, which will turn on, change... It’s a good thing, though! Viral delivery has several advantages over other mechanisms. For one, many...permanently integrate and/or express long term (up to several years!). Biosensors tend to be more reproducible...

...more effectively used in a technology or as a therapeutic.  A (very) brief history GCE came onto the synthetic...specific to the ncAA and orthogonal to (i.e. non-interacting with) the cells’ own natural translation machinery...system (including media, cell type, and growth temperature) may need to be individually optimized for each...focused on turning GCE tools developed in the literature into robust and universal techniques that researchers...fulfill their mission. While they’ve shortlisted several protocols, including their phosphoserine ncAA protocol...., Griffith, M. C., & Schultz, P. G. (1989). A General Method for Site-specific Incorporation of Unnatural...

...synthetic) pFZ19 (yellow flame) Temperature inducible promoters Temperature sensitive expression systems ...Promoters control the binding of RNA polymerase and transcription factors. Since the promoter region ...Types of inducible promoters Chemical agents, temperature, and light are all examples of factors that can...promoters; they show near-zero expression at regular temperatures but can be induced by heat or cold exposure....since expression can be repressed by glucose. Temperature inducible promoters are also known for their ...

...interest, as it is prone to generating indel errors. Indel errors generated in the course of repair by ...vast toolbox of processing factors, including polymerases (Pol μ and Pol λ), nucleases (Artemis), and structure-specific...illustrated below, NHEJ-mediated repair of Cas9-generated breaks is useful if the intent is to make a knockout...overhangs is available. Cas9 can also be altered to generate a targeted single strand break; when two such ...Chromosomal Translocations in Human Cells Are Generated by Canonical Nonhomologous End-Joining. Molecular...

...PubMed PMID:29483642. Making protein-protein interaction studies easier with a 2in1 vector cloning system...cloning system is well suited for protein-protein interaction (PPI) studies in plants that rely on transient...Grefen Lab has utilized the 2in1 cloning system to generate a series of plasmids to study PPI using Förster...Plasmid Toolkit consists of 4 plasmid toolkits generated with 4 different, binary 2in1 vectors with all...powerful approach to control protein-protein interactions in cells because the protein binders can be ... that these photo-reversible protein-protein interactions can control subcellular localization processes...

... moment that Teytelman realized that running interactive workshops to train researchers on tools and best...next steps. April Clyburne-Sherin had already run several reproducibility workshops as Director of Scientific...to this open science collaboration. The first iteration of these workshops took place at Caltech in Jan...being adopted by a wider group of researchers. Generating material to empower others to practice and teach...team was assembled when it was realized that interactive workshops that train and provide researchers ...on the Addgene blog: Check out this guide on Transferable Skills: External Collaborations Learn about ...

...a high imidazole buffer that outcompetes the interaction between the protein of interest and the metal...polyhistidine tagged protein of interest (purple) and several contaminating proteins (blue, green, orange) flows... low imidazole wash buffer disrupts the weak interaction of the contaminating protein but not that of ...A high imidazole elution buffer disrupts the interaction of the protein of interest and it is successfully...protein conformation, the polyhistidine-metal ion interaction is not disrupted by denaturants (Hochuli, 1988... of a protein. Neither the N or C terminus is preferable over the other however, tag placement can affect...

...way they are, the research antibodies in your refrigerator often need a little help to be useful. Mainly...enzymes. The most common enzyme conjugates are Horseradish Peroxidase (HRP) and Alkaline Phosphatase (AP...This chemical coupling can be achieved through several different mechanisms. Most take advantage of chemical...sequence or that rely on strong protein-protein interactions with other peptides, like an Fc-binding domain...acids or peptide tags. 5) Strong protein-protein interactions with other peptides. Created with BioRender....Ligation Assay for Detecting Protein-Protein Interactions and Protein Modifications in Cells and Tissues...

... only for translational research, such as gene therapy, but also basic research. To improve cell-type ... with high rates of labeling are ideal of gene therapy applications, promoters that produce sparse labeling...makes it easier to trace a neuron’s projections. Generating animal models: Transgenic animals allow for the...tissues, but they are costly and time-consuming to generate, especially for large animals such as primates...basic as well as translational research for gene therapy. The Roska lab hopes to design improved versions...blog Read more about using AAV for retinal gene therapy Find more about precise neuronal targeting using...

...prep is straightforward and rapid as there are several commercially available p24 ELISA kits that can ...plasmid was used in the transfection; if preparing several different lentiviruses in parallel, this approach...expansion, and confirmation. This procedure can take several months but should be considered in labs that will...virus or require precise titers. Additional considerations for titering lentivirus Volume and transduction...4.4 (2009): 495-505. PubMed PMID: 19300443. 2. Geraerts, Martine, et al. "Comparison of lentiviral vector...predicting gene transfer events." Genetic vaccines and therapy 2.1 (2004): 1. PubMed PMID: 15291957. PubMed Central...

...interesting discoveries. You’ve analyzed the data and generated beautiful figures. And, you can’t wait to tell...need to first make your poster. While a poster generally contains the same sections as a primary research...references and contact information, and it is generally acceptable for the font here to be much smaller...is better! Your poster should be readable from several feet away. It can also be useful to use two different...it’s good to let your ingenuity shine, and you generally have free reign over how you put your poster together... as rows. It can be more catchy because it is generally less common and will stand out; however, be careful...

...and their use for HDR editing and mouse-model generation. Key components of Nanoblades Nanoblades are ...hematopoietic stem cells (HSCs) using Nanoblades. Overall, editing efficiencies were between ~50-70% for ...lower rates of off-target effects. Nanoblades generated up to 76% editing efficiency in mouse BM cells...-target effects, with electroporation of RNPs generally having lower off-target edits compared to DNA ...gRNA, Cas9, and a HDR donor template DNA can be generated by incubating Nanoblade particles with polybrene...Nanoblades are a potential alternative method for generating transgenic mice strains and for non-invasive ...

...them to see how feasible they were, conducting literature reviews and having weekly journal clubs outside...this process, we had an idea that survived the literature review, survived technical feasibility analysis...evenings on the project, but still continued to generate new ideas as they popped into our heads. Anything...color change until a few months later. RevBio accelerates with help from SynBio Axlr8r We limped along ...business. We ended up applying to a business accelerator freely stating that we had an idea, knew how ...rough day, and we have great partners in this accelerator program who want us to succeed. We’re excited...

...DNA, the CTD is also sometimes called the PAM-interacting (PI) domain. The guide RNA is bound primarily...most likely through unstructured electrostatic interactions. It also can’t recognize PAMs, so it associates... less stringent recognition mechanism that can tolerate mismatches in this region. The crystal structure.... As mentioned above, however, Cas9 sometimes tolerates mismatches in the next 8-10 bases after perfect...this region of Cas9 also allows the protein to tolerate RNA:DNA mismatches between base pairs 18 and 20...Each new structural insight also spurs the next generation of engineered CRISPR enzymes with their own tradeoffs...Molecular graphics and analyses performed with UCSF ChimeraX, developed by the Resource for Biocomputing, Visualization...

...the conference After using your elevator pitch several times at the conference, you might notice some ...larger side? At large conferences, chances are that several talks will be going on at once in different rooms...a conference, be sure to practice beforehand. Generally, two types of people will visit your poster: those...have “tweep ups” where you can meet those you’ve interacted with on science Twitter in real life. If there...the tweep up (and for the entire conference, in general). After the conference Review your favorite conference... considered networking, but here are some more general tips. Before the conference Networking is key even...

...that Mobile-CRISPRi is transferable into multiple bacterial species including several involved in human disease...and is stable and active for greater than 50 generations. Figure 1: Mobile-CRISPRi's modular components...highly conserved glutamine-fructose-6-phosphate aminotransferase (glmS) gene in the recipient bacteria due ...Mobile-CRISPRi to knockdown individual genes in several bacteria associated with human disease (S. aureus... library strains was measured after 6 and 12 generations with or without induction of CRISPRi knockdown...

... (cap) genes. AAV replication uses the host’s polymerase, but requires Rep proteins to process a double-stranded...consuming to prepare the 40% PEG solution. PEG takes several hours on a heated stir plate to dissolve and requires...carefully layered and then the viral suspension generated during AAV production is overlaid. Following ultracentrifugation... comparing to an AAV standard curve, which is generated from an ITR-containing plasmid. This whole process... always be included. While the physical titer generated by qPCR is useful, I test a few different concentrations...inverted terminal repeat sequences. Human gene therapy methods, 23 1, 18-28. PubMed PMID: 22428977. 2....

... copy number variation (CNV) assays. There are several varieties of these, but in this blog post, we're...length of the amplicon. As the probes can have several different markers, it can also be used to run multiplex...Do you need to call exact copy numbers or just general trends? How confident do you need to be in your...Addgene blog   Droplet Digital PCR for AAV QuantificationPolymerase Chain Reaction Overview and Applications...Applications Resources on Addgene.org Protocol: Polymerase Chain Reaction (PCR) Protocol: How to Design a primer...SYBR Green I and TaqMan quantitative real-time polymerase chain reaction methods for the determination ...

...incubation at 55 °C - a dangerous, non-physiological temperature for mammalian cells and one which diminishes ...could detect knock-in of tags at a target site. Overall, 64% of researchers were unhappy with the new method...the water. On the bright side, successful indel generation and epitope knockin was achieved by a few respondents...ssDNA leading to inefficient downstream editing. Overall, the real take-home message thus far, based on ...form a complex with a 5’-P-ssDNA guide at room temperature. Until that time, those who have been lucky with...Pooran Dewari is a postdoc at the MRC Centre for Regenerative Medicine (CRM), Edinburgh. His research interests...

...experiments require some consideration of FP properties. It is important to look out for general problems associated... judgement by simply “looking at them”. Modern cameras and image analysis software are highly sensitive...microscopy cannot be interpreted as direct molecular interaction without further investigation. One fluorescent...microscopy method commonly employed for analyzing interactions between two molecules more closely is Förster...PMC4777285. Further Reading Microscopy U. Generfal Considerations when Using Fluorescent Proteins. Ruusuvuori...