...and are often impractical for small projects. Most researchers instead turn to so-called “mismatch nuclease...involves splitting up a sample into thousands of physically isolated partitions (such as droplets or wells...Since there are many thousands of partitions, statistical modelling can be used to determine the actual..., does not require standard curves, and can theoretically detect a single target molecule in a sample....wild type target in each sample can be quickly calculated (details in [2]) and can be used to quantify ...mutations versus those with the bi-allelic mutations typically desired when screening clones for complete functional...

...systems are available at Addgene! Chemically inducible promoters Chemically regulated promoters are among ...sensitive expression systems are typically less leaky than chemically induced promoters; they show near-zero...transcription is turned ON. Types of inducible promoters Chemical agents, temperature, and light are all examples...decreasing promoter leakiness.   Other examples of chemically induced promoters include positive inducible ... promoters only work in prokaryotic cells and typically only in the same or closely related species from...

...emerging engineering technology. Monoculture and chemical spraying, mainstays of modern agriculture, are... by developing a plant biotechnology product specifically for consumer sales. Flowers that change color...a new conversation about GMOs.  Challenge #2: Technical obstacles to plant synthetic biology In addition...understanding of plant biology is a significant technical obstacle to plant synthetic biology. We cannot...creative problem solving like the Howard Hughes Medical Institute have begun to pivot towards plant biotechnology...organism for plant research for decades, and the biochemical pathways involved in central anthocyanin metabolism... – our project wouldn’t be possible. A second technical hurdle is the lack of resources associated with...

...mammalian cells often relied on chemical (CaCl2, cationic liposome) or electrical (electric current forming ...research since their first use in the early 90’s to genetically modify primary cells. Amongst the different vectors...its safety was a disadvantage particularly for clinical development. In addition, MoMLV is inefficient...lentiviral vectors was developed for safe use in a clinical setting. In this generation, the HIV Tat gene ...lentiviral vectors can be used for research and for clinical purposes, however improving the safety of these...than with the 2nd generation. Knowing this you can scale up your production set-up and purify and concentrate...virus in order to aid viral binding. This method, called spinoculation, has been found very effective for...

...HE, Lee C-YS, Calidas D, Lu T, Smith J, Schmidt H, Krause MW, Seydoux G (2014) Scalable and Versatile ...is an assistant professor at the University of California, Santa Cruz. Emerging CRISPR/Cas9 editing technologies...melting and rapidly cooling dsDNA repair templates dramatically boosted editing efficiency (Ghanta et al., 2020... more efficient. Pure PCR repair templates are critical for efficient knock-ins (Ghanta et al., 2020)....Farboud et al., 2019). Work from the Mello lab has dramatically improved RNP editing with linear repair templates...is an assistant professor at the University of California, Santa Cruz where his group focuses on gene regulation...Tzur YB, Friedland AE, Nadarajan S, Church GM, Calarco JA, Colaiácovo MP (2013) Heritable Custom Genomic...

...pathways and how they lead to neurological phenotypes observed.” Using genetically-encoded biosensors and iPSC-derived...Scientific Advisor. It’s possible that solute channels typically expressed on other cell types can compensate for...understand so that we could possibly develop pharmaceutical interventions that would inhibit IL-36 release...oligomerization, signalling complex assembly, and localization to the nucleus,” says Dr. Terry Hébert, a professor...professor and assistant dean of biomedical science education at McGill University.  These protein-receptor... iPSC-derived cortical neurons from patients with GNB1 mutations, the lab hopes to capture the signalosome...

... The previous post in this series described a practical approach to selecting a bright fluorescent protein...amounts of excitation power (Shaner et al, 2008). In confocal laser scanning microscopy, fluorescent proteins...selective plane illumination, TIRF or spinning disk confocal use entirely different excitation power regimes...performed with soluble fluorescent proteins or localized fusion proteins and do not require dedicated plasmids...Amsterdam). He develops, characterizes and uses genetically encoded fluorescent probes. You can follow him...(University of Amsterdam) where she develops genetically encoded FRET based biosensors.     Additional...Fluorescent Tags Find Plasmids for Subcellular Localization Browse the entire Fluorescent Protein Collection...

...Therefore, it is not possible to visualize the localization of protein synthesis. Alternatively, fluorescent...graduate studies I attended a scientific symposium called Horizons in Molecular Biology. This annual symposium...PhD students at the Max Planck Institute for Biophysical Chemistry, Göttingen in Germany. I attended this... symposium seeking inspiration to resolve the technical hurtle of monitoring protein turnover of specific...interesting talk by Prof. Erin M Schuman, a former Caltech researcher, now Director at the Max Planck Institute...technique applies metabolic labeling using a noncanonical amino acid, azidohomoalanine (AHA). In the technique...application of AHA labeling is known as fluorescence noncanonical amino acid tagging (FUNCAT, Dieterich et al....

...simulate physiological gene expression regimes that cannot be easily achieved with classical gene overexpression... super-resolution microscopy.” Endosomes are historically difficult to study in living cells using conventional...system to study epigenetic modulation. While catalytically inactive cas9 (e.g. dCas9) has been fused to...activate gene expression, sometimes incorporating chemically inducible approaches to provide temporal control...kinetics of chromatin regulation and subsequent mathematical modeling. Its reversibility allows precise and...hER or progesterone for hPR - leads to nuclear localization, binding to the appropriate promoter sequence...

... the flick of a light switch. Neurons are not typically persuaded to fire when light is shined on them...channelrhodopsins. Their new channelrhodopsins, called ChRgers, make the brain a little easier to access...determine membrane expression and plasma membrane localization of transmembrane proteins, like rhodopsins, ...directed evolution, because channelrhodopsins are typically evaluated with low-throughput methods such as ...properties of a channelrhodopsin, such as cell localization, kinetics, and spectral properties, must be ... midbrain, fiber-optic cannulas were still stereotaxically implanted into the mice’s brains so that light...optogenetics.  TL;DR The new channelrhodopsins, called ChRgers, make it easier and less invasive to use...

...likely that the shifts caused to the practical and philosophical landscape of the scientific enterprise...precipitated increases to the NIH recommended pay scale – used by many institutions to set postdoc wages...focus toward advocacy and action with sessions specifically chosen to better define productive routes for...Riglar. David T Riglar is a postdoc at Harvard Medical School. In the lab, he uses synthetic biology to...References: 1. Alberts, Bruce, et al. "Rescuing US biomedical research from its systemic flaws." Proceedings...Pubmed Central PMCID: PMC4304227. 4. Revitalizing biomedical research: recommendations from the Future of ...

...visualizing ncRNA localization and function, but directly incorporating very long ncRNAs is impractical. Instead...the full ncRNA locus (Ho et al. 2012). For large-scale screening, sgRNA libraries are useful for targeting...short when it comes to targeting precision and practical application. If the CRISPR/Cas9 system is not ...interact with specific chromatin structures or other local genomic elements (Hazan and Bester 2021). Other ...can cleave single-stranded RNA using gRNAs to specifically edit or degrade targeted lncRNAs. The Cas14 ...15476286.2021.1899500 Engreitz JM, Haines JE, Perez EM, et al. Local regulation of gene expression by lncRNA promoters...

...are commercially available. Titer calculation (1 or 10 days) Physical titer (particle count, rAdV genomes...repeated multiple times (2-4 rounds) at increasing scale over the course of 1-2 weeks. Each round of amplification...genomes/mL) can be measured by: Optical density: particle concentration is measured by looking at absorbance...260nm corresponds to 1.1 x 1012 particles/mL. To calculate the particle number: OD260 reading x dilution ... days later. Plaques are counted and titer is calculated in terms of plaque forming units (pfu), taking...1236. PubMed PMID: 17546019. 2. He TC. et al. A practical guide for using the AdEasy System. 3. Jager, Lorenz...

...mass spectrometry (AP-MS), lack the ability to specifically identify proteins that interact with a particular...lysed before the bait protein is pulled down, typically with an antibody. This can artificially select...controllable version of the BioID protocol. The critical component for both BioID and Split-BioID is BirA...functional BirA* enzyme. This type of experiment is called a protein fragmentation complementation assay (... via Western blot, and that result in proper localization of the fusion protein. Advantages of Split-BioID...Further characterization of these candidates is critical to understanding how they interact with the bait...Varnaitė, R. (2016). Meet the neighbors: Mapping local protein interactomes by proximity‐dependent labeling...

...designed by synthetic biologists to perform large scale, one-tube cloning reactions. In this blog, we’ll... Gate cloning is MoClo. That’s because MoClo specifically refers to the organizational schema described...organization schema MoClo can be broken down into three hierarchical levels: individual DNA parts, transcriptional...together. They can express one or more gene(s), and typically begin with a promoter and end with a terminator...sounds really neat — but when is this type of large-scale assembly useful, you may ask? You might be surprised...microbially or in cell-free systems. These pathways typically involve multiple steps in which a precursor molecule...and subsequent necessary modification of these chemical products. The flexible nature of MoClo allows ...

...generated in vitro using synthetic genes that are typically expressed from a plasmid or from an integrated...share, making recombinant antibodies the most practical choice. Hybridomas are much tougher to ship, often... often lack sequence data, and may genetically drift over time. Antibodies made from animals are limited...the ease with which scientists can now improve genetically-encoded research tools, it is shocking that the...Begley CG, Ellis LM (2012) Raise standards for preclinical cancer research. Nature 483:531–533 . https:/...Crosnier C, Staudt N, Wright GJ (2010) A rapid and scalable method for selecting recombinant mouse monoclonal...for the Selection of Monoclonal Antibodies for Clinical Use. In: Antibody Engineering. InTech Additional...

...science, dating back several hundred years, with historical ties to psychology and philosophy (the mind-body...chemogenetic tools are designed channels that are chemically activated. Both are introduced in neurons and...modifications at the DNA level in a temporally and locally defined manner. This allows scientists to draw ...and not targeted to a specific area. Some other technical considerations for stereotaxic injections are:...be high enough to maximize possible outcomes. Typically, AAVs have to be minimum around 1012-1013 vg/ml...has to be large enough to target larger areas, typically a few hundred nl. Alternatively, for larger brain...expression levels of the viral vector trangenes typically occur two weeks after injection. Therefore, it...

...embryos. But once you generate a growing colony of genetically engineered mice, what can you do? In this post...Cre-lox lines, and how to properly control for genetically engineered mice in your experiment. As you’ve...ve learned in Part 1, there are many types of genetically engineered mice: transgenic mice, knockin and...getting a small piece of tissue from each mouse (typically via either a blood draw, tail clip, or toe clip...Hemizygous mice can be included in breeding strategies identical to those used for breeding heterozygous mice. ... cases Whether your mouse colony consists of genetically engineered mice with a single mutation or strains...prepared to plan an experimental strategy that uses genetically engineered mice. Best wishes on your experiment...

...What’s behind these sights are an abundance of biological molecules like chromoproteins, fluorescent proteins...proteins and luminescence have been widely used in biological research for a long time, chromoproteins are ...just now being developed for a broad range of biological inquiries.  What is a chromoprotein and how ...photosynthetic activity. This still allows photosynthetically active wavelengths to reach zooxanthellae...the Sea AnemoneCnidopus Japonicus. Journal of Biological Chemistry 281:37813–37819 . https://doi.org/10.1074...Pigments of Pocilloporid and Acroporid Corals. The Biological Bulletin 189:288–297 . https://doi.org/10.2307...chromoproteins for bacterial synthetic biology. Journal of Biological Engineering, 12(1). https://doi.org/10.1186/s13036...

...reverse-transcribed piece of DNA called a “provirus.” This DNA is synthesized inside of what is called the “target cell...The Retroviridae (commonly called retrovirus) family — of which HIV is a member — may seem like an unlikely...retroviral vectors and 2.) a subclass of retroviruses called the lentiviruses (like HIV-1) — that have been ...(Prussin et al., 2015), only a small fraction typically cause disease. The gamma-retroviruses have their...transduction by vectors (which we sometimes still call “infection” as a shorthand, although it is not quite... proteins encoded by complex retroviruses. Microbiological Reviews, 56(3), 375–394. https://doi.org/10.1128...