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... This means recombinant proteins can aggregate or precipitate out as insoluble granules. We overcame this...decidedly uncomfortable 45°C). Their more adventurous cousins are the extremophiles - the legends who not only...of processes. The food processing industry, for instance, employs psychrophilic enzymes for milk processing...enzymes for the host The expression of recombinant proteins for industrial or research purposes requires stable... standard conditions. However, one of the many proteins which are not compatible with regular mesophilic...temperatures suitable for the expression of psychrophilic proteins. The primary problem we wanted to overcome was...folding and expression of recombinant psychrophilic proteins. As the figure below shows, E. coli expressing...

...possible to generate antibodies against many types of molecules including proteins, lipids, and carbohydrates... post we’ll focus on antibodies that recognize proteins. Figure 1: Steps in the generation of hybridomas...hybridomas come from animals that were immunized against the target protein. Animals are first injected ...special medium called HAT. This medium selects against unfused myeloma cells and antibody-producing B ... will exhibit off-target binding to unintended proteins. This is because monoclonal antibodies recognize...amino acid sequence will be shared across other proteins. Conversely, polyclonal antibodies - which are...recognize epitopes that are also present in other proteins. Because of the specificity of monoclonal antibodies...

...podcast segment The human proteome contains a breadth of modular domains that facilitate protein-protein ...for RNAi. The Vellai lab’s new plasmid, T444T, contains T7 terminators adjacent to the T7 promoters to...TurboID and miniTurbo Article contributed by Eric Perkins  Listen to the TurboID and miniTurbo podcast segment... study of the distribution and interactions of proteins within these structures. Enzyme-catalyzed proximity...distribution and interaction networks of specific proteins in living cells. Until recently, the enzymes most...APEX2 and BioID. APEX2 is fast, tagging proximal proteins within minutes, but requires H2O2, which can be... tailored for the detection of other norovirus strains and even other viruses by modifying the sequence...

...majority of Type VI-D loci contain accessory proteins with WYL domains (named for the three conserved amino acids...domain-containing proteins may be broader regulators of Cas13d activity. This property makes WYL proteins an intriguing... near a putative effector nuclease. The Cas13d proteins they identified have little sequence similarity...Cas13a-c orthologs, but they do include HEPN nuclease domains characteristic of the Cas13 superfamily. Yan et...advantages, including Cas13d-specific regulatory proteins and high targeting efficiency, both of which are...intriguing counterpart to anti-CRISPR proteins that negatively modulate the activity of Cas enzymes, some...specific editor in mammalian cells Not all Cas13d proteins are functional in mammalian cells, but Konermann...

...available here for free!) contains strategies, hints, anecdotes, and insights. After I wrote the first ... at the National Institutes of Health, was an Associate Professor at Albert Einstein College of Medicine...Hughes Medical Institute. His interests include the quality control of secretory proteins in the Endoplasmic... and mentoring to several people that found my insights helpful. All went on to get faculty positions ...specific questions and experiments. Nobody EVER complains that a seminar was too easy to understand. Make... Distinguishing correctly folded and misfolded proteins is essential for human health. Failure to do so...terms. "Implications for human disease" is vague. Instead, consider "A mechanistic understanding of this ...

... HIV so that infected cells will produce viral proteins and die, either through cytotoxicity or the immune...transcription, but with two additional activation domains (MS2 and p65) to enhance transcriptional activation...expression. In cell lines that can produce HIV-1 toxic proteins, CRISPR/Cas9 SAM caused apoptosis, indicating ...cell and 2. prevent reinfection of those cells. Kaminski et al. designed gRNAs to target the HIV-1 5’ and...Importantly, no off-target effects were detected. Kaminski et al. observed no negative effects on cell viability...up to 7 mismatches to the gRNA target sequence. Kaminski et al. next tested their procedure in HIV-1 infected...expanded, and infected with HIV-1. For two HIV-1 strains, Cas9/gRNA lentiviral expression significantly ...

...composed of four peptide chains (two identical heavy chains and two identical light chains) each with a constant... The chains are covalently linked to one another by disulfide bonds between the two heavy chains and between...are composed of just the variable domains of the heavy and light chains of an antibody. Arguably not a “...chain and one light chain. When assembled, the four chains make a Y-shaped protein that can be divided into...portions of the constant regions of the two heavy chains. In the lab, this is the region that determines...fragments are produced by proteolytic digestion that retains an antibody’s disulfide bonds within the hinge ...the fragment could bind not only the endogenous proteins you are trying to block but your primary antibody...

...endogenous proteins is an exciting strategy, it is difficult to apply this technique to oncoproteins that lack..., a postdoctoral fellow at Dana-Farber Cancer Institute. Targeted protein degradation In the Bradner and...synthesize compounds that enable selective removal of proteins-of-interest from the proteome. Rather than inhibiting...degraders” recruit the proteasome to destroy targeted proteins. We previously developed small molecule degraders...that achieve selective degradation of endogenous proteins (notably, BRD2/3/4, CDK9, TRIM24, FLT3, BTK, and...linking small molecules that bind these target proteins to other small molecules that bind an E3 ligase... molecule) and expression of FKBP12F36V-tagged proteins. The dTAG molecule induces ternary complex formation...

...these three kinases, photoswitchable Dronpa domains were inserted at sites homologous to the locations they... other kinases and proteins. Zhou et al suggest that more photoswitchable proteins could potentially be... be created by inserting pdDronpa domains into loops of a protein of interest. If you plan to try this...ninja term for vanishing, and pa= photoactivation) domains on either side of the active site of a constitutively... Learn More about Photoactivatable Fluorescent Proteins Control Protein Expression with Cre-lox Control...reporter plasmids Find photoactivatable fluorescent proteins in empty backbones in the Michael Davidson Collection...Photoswitchable Fluorescent Proteins ...

...transversion editors. New DNA repair proteins fused to CGBEs Instead of using UNG, the Wei Leong Chew Lab...repair proteins fused to CGBEs The David Liu lab also experimented with adding different proteins to the...approach to improving C → G editing. Instead of fusing DNA repair proteins to the base editor, they added the...inhibit BER during cytosine-to-uracil editing, and instead introduced its opposite, a uracil DNA N-glycosylase...excising the freshly edited U bases — but E. coli instead filled those abasic sites with A's, producing C...Mycobacterium smegmatis, termed UdgX, they found three proteins whose fusion improved the CGBE: DNA polymerase...(RBMX) (Koblan et al., 2021). Several of these proteins had additive effects when multiple were included...

...fluorescent proteins system for Caenorhabditis elegans By Jennifer Tsang Fluorescent proteins (FPs) traditionally...Tools for labeling excitatory inhibitory synaptic proteins Fluorescent protein system for C. elegans Fluorescent... Labeling excitatory and inhibitory synaptic proteins with FingRs By Jason Nasse Since the pioneering...neurotransmitters. Using genetically encoded fluorescent proteins coupled with Fibronectin intrabodies generated...FingR constructs target the synaptic scaffolding proteins PSD95 and gephyrin to label excitatory and inhibitory...neuron, the FingRs bind to the endogenous synaptic proteins allowing them to localize to the synapse and minimize...combining vectors expressing different fluorescent proteins targeting inhibitory or excitatory synapses or...

... process. When using zinc finger proteins or TALE proteins, proteins had to be re-engineered for each ...that bind to MS2 proteins. These MS2 proteins then recruit additional activation domains (HS1 and p65). ...transcriptional activators: some methods rely on fusion proteins while others re-engineer components of Cas systems...method is its construction: it relies on antibody chains, which are relatively large and are not expressed...epitope tag of SunTag with the P65-HSF activation domains used in SAM. This method has been shown to activate...Davidsohn N, Housden BE, Perrimon N, Weiss R, Aach J, Collins JJ, Church GM (2015) Highly efficient Cas9-mediated...Kowal EJK, Buchthal J, Housden BE, Perrimon N, Collins JJ, Church G (2016) Comparison of Cas9 activators...

...images of proteins. But they are still flat. To truly appreciate the 3D structure of these proteins, it is...Molecular Designs, we create physical models of proteins using 3D printing technology. The tagline for ...to create stunning, interactive images of their proteins. Since the 1980’s, journal articles have been ...structure? We have been creating physical models of proteins for researchers for the past fifteen years. On...expressed purpose of creating physical models of proteins and other molecular structures for use in professional...Under the guidance of a trained educator, these instances of focused attention become magical teaching moments...their research while creating physical models of proteins that are being investigated in their labs. Soon...

...elements anymore. The kit contains sequences for several different fluorescent proteins. Visualizing fluorescence...Nicola Patron is Head of Synthetic Biology at the Sainsbury Laboratory, where she often feels more like an... between plants and their pathogens. As Patron explains it, her work has always been focused on gene transfer...Cloning (MoClo) Kit. What does it include? Patron: Sainsbury has a mandate to share and educate and enable ...chlorophyll, so we looked at newer fluorescent proteins that get around that problem. MoClo is really ...straightforward really and we’ll be producing an instructional video soon to make it easier for people to use...for plants? Patron: Our lab and others at the Sainsbury are working on a lot of CRISPR tools for editing...

...forces its insect host to dive into water. There, the worm emerges to find a mate, leaving the insect dead ...infected grasshoppers expressed different proteins in their brains compared to uninfected grasshoppers, including...Plasmid pumpkins, team costumes, and spooky science stories. Yes, please! At Addgene, we love finding...addition to the Halloween contest, we’ve also carved pumpkins (yes, Blugene made an appearance there too) and...base of the tongue where it pierces the tongue and drains all the blood out. Once the blood is depleted, ... the tongue atrophies and falls off. The louse remains becoming the fish’s new “tongue” (Brusca and Gilligan...Spinochordodes tellinii‘s larvae makes its home inside grasshoppers and crickets. When it’s ready to transform...

...lentivirus, but instead is contained by a capsid which is comprised of the viral proteins VP1, VP2, and ...Maday et al., 2014). It is currently unclear which proteins facilitate transport of neuronal tracing compounds...peroxidase (HRP; Kristensson and Olsson, 1971), plant lectins (wheat germ agglutinin WGA, Schwab et al., 1978...iontophoresis (applying small electric currents via inserted electrodes) may be applied, but these methods ...neuroanatomy and neural connectivity, providing valuable insights for neuronal communication. Viral vectors and...methods such as electrophysiological recordings. The origins of these methods are naturally occurring viruses...engineered to have differences in their capsid proteins. For a brief overview see Addgene’s AAV guide....

...tweet URLs with scientific domains <10% of the time. Figure 3: Top 20 Domains Tweeted by Scientists. 6....scientific domains more often than other scientists   Figure 5: Portion of Tweets with Scientific Domains, Categorized.... The #1 (re)tweeted domain by scientists was Instagram Social media and news sites were also popular....6. The top scientific domains tweeted by biologists were: Nature, Sciencemag, Wiley, Cell and Scientific...Scientific American Figure 4: Top Scientific Domains Tweeted by Biologists. 7. Biologists, neuroscientists ...(orange box) post tweets containing scientific domains more often than other disciplines. The majority...tweets for most disciplines contain scientific domains between 0-10% of the time; however, ~20% of life...

...editor can install all possible point mutations, delete short stretches of DNA and even insert new DNA at...advancements and the noteworthy work of fiction that it inspired. CAUTION: Spoilers alert! Pre-DNA Era  H.G. ...enzymes, meganucleases, were discovered. These proteins are characterized by large DNA recognition sites... successful in the box office, indicating the insatiable appetite of the moviegoers for dinosaurs wreaking... In this movie Ethan Hawke’s character revolts against class divide that is created by genetics.    Zinc...DNA-cleaving strategy similar to the ZFNs, but instead of recognizing nucleotide triplets, the components...target new genomic sites. The discovery of the Cas proteins in 2005 was a major breakthrough in the world ...

...encoding the insert of interest as well as sequences necessary to encapsulate that insert into a viral... psi signal, the genes encoding the structural proteins could not be integrated into the new viral particles... one with the exception that the HIV accessory proteins that are not essential for the production of the...its activity. A CMV or an EF1α promoter has been inserted in this LTR so you don’t need Tat to transcribe...RCL) Efficient gene transfer Potential for insertional mutagenesis: Replication-incompetent lentiviruses...human tropism can still infect the researcher and insert into her/his genome - this is a biosafety risk ...dividing and non-dividing cells No immunogenic proteins generated Stable integration into host genome...

...Director of the Merkin Institute and Vice-Chair of the Faculty at the Broad Institute; Professor of Chemistry...Harvard University, and David R. Liu from the Broad Institute and Harvard University. Over the course of the...papers (data was provided by Addgene on 5/26/20 and contains requests from 5/22/14 to 5/19/20). Each paper ...data with a 7-day rolling mean gave us a crude insight into the magnitude of the effect COVID-19 had on... analytical expression for the data likelihood contains an intractable integral of the product of a Poisson... least impacted. These results highlight the instability and uncertainty in estimating parameters from...important additional caveat is that our dataset contains plasmid orders from just one lab, which limits...