...co-coregulator) partners. The coregulator ‘core’ complex is composed of a tightly-bound invariant group of proteins...coregulators exist as multiprotein complexes, a member of a single coactivator complex can serve as a rate-limiting...seeking to compliment these consortium-generated datasets with a systematic effort to compile NR- and coregulator-related...physiological processes are controlled by deliberate and compulsory actions instigated by the central nervous system...factors found in metazoans. The superfamily is comprised of 48 human and 49 murine members. NRs permit ...metabolic, and immune response programs. NRs also encompass one of the most successful targets for drugs currently...interact directly with NRs) exist in steady-state complexes with secondary coregulator (or co-coregulator)...

...iPSC cell lines, which can complicate comparisons made between lines. Incomplete silencing of transgenes ...are prone to incomplete silencing of reprogramming transgenes, which leads to incomplete reprogramming... heterogeneity between clones. This can complicate comparisons made between samples, i.e. transgene expression...efficiency of reprogramming when compared to other methods, even when compared to integrating viral approaches...less time to complete. Morphology is a simple and fast to evaluate. iPSCs should form compact colonies that...and have a higher efficiency of generating iPSCs compared to other delivery methods. Retrovirus has a cloning...lentivirus has similar rates of reprogramming when compared to MMLV-derived retroviruses, it is not as well...

.... The team at the Victoria Makerspace is composed of computer scientists, engineers, and biologists working... elegans as a computer model. When I first heard about it, I got excited from a computer scientist perspective...defects above 25 degrees. Computer scientists and biologists work together to compare the predictions about...interests in biology and computing. In 2009, he founded a team at UVic to compete in iGem, an MIT-sponsored... really epitomizes what the makerspace is. The computer scientists can get involved in modelling, the ...Developing these skills relies upon biologists and computer scientists learning and communicating with one...coding the molecular biology of the worm into a computer model that recapitulates how molecular biology...

...of oxidation (dehydrogenation). MitoTimers are composed of a Timer protein fused to the mitochondrial ...upregulate an alternative pathway or induce other compensatory mechanisms. How can protein function be dissected...’s lab, acutely depleted proteins from their compartment of action via sequestration. The strategy relies...outer mitochondrial membrane. Sequestration is accomplished by knocking down your endogenous protein of ...May 1;126(Pt 9):2102-13. Assemble multiple component plasmids from building blocks via Golden GATEway...GATEway Cloning Frustrated by the complexity of assembling recombination of transgenesis constructs? A new...cloning kit simplifies the cloning process for complex DNA constructs, particular for those involving ...

...different compositions of the Cascade complex. Created with BioRender.com.   The Cascade complex Cascade... an important component of the CRISPR Cas3 system. Most Cascade complexes are comprised of a combination...systems work using a multi-Cas complex called Cascade (CRISPR-associated complex for antiviral defense). Cascade... Cascade complex from CRISPR type I-E. Created with BioRender.com. Thanks to this complex, developing... It can be difficult to ensure comparable expression of all components of Cascade for maximum efficiency...Cascade-Cas3 plasmids in Addgene's collection! A “complex” system Cas3 is part of the Class 1 CRISPR family...from different species and will have different compositions of Cascade. Although seemingly ubiquitous in...

... pre-mRNA splicing in a compound screen. Their reporter system is comprised of two plasmids: Luc (intronless... Vacuolar Compartment mCherry-Rab5 BFP-Rab5 GFP-Rab5B Vacuolar & Budding Compartment GFP-Rab4B ...insights into the make-up of multiprotein complexes like the EPC1 complex.. Going one step further, they also...about it! Split Cas9 system The Cas9 protein is composed of an n-terminal DNA recognition domain and a ...sequencing or other readout. Cellular memory is accomplished by the use of sequence-specific enzymes (e.g...calculated (the BRET ratio), which allows for compensation of varying fusion protein expression levels....more powerful LID, the authors recently used computational protein design, phage display and high throughput...

...important that the digest go until completion. The time required for complete digestion varies for different...want to use electro-competent cells instead of the more common chemically-competent cells. You should perform...fragment of DNA (insert) that has been cut by compatible restriction enzymes. An enzyme, DNA ligase, then...plasmid to the new fragment thereby generating a complete, circular plasmid that can be easily maintained... are cut with restriction enzymes to generate compatible overhangs that allow them to bind. Ligase is ...your backbone and insert - these both must have compatible cut sites for restriction enzymes that allow ...    Alternatively, this whole process can be completed using a single enzyme if your insert is flanked...

... work for biotechnology companies and help other researchers use the company’s products successfully. ...guidance, and resolve complaints about products. As you might expect, different companies produce different... duties. Bigger companies have dedicated technical support teams, sometimes comprised of several layers...cells (manufactured by one of the larger biotech companies), and as far as I remember, it was the first time...time that I was absolutely convinced it was the company’s fault. I contacted them to express my concerns...rather painful to get there: I had to send the company detailed documentation of my results, answer loads...takes some time (several years) to build up comprehensive lab experience. With this valuable training ...

...lead to comparable experimental results. Using PiggyBac transposons, the Woltjen Lab compared different...robust engineering. These components are provided in Golden Gate compatible backbones that can be combined...latest hot plasmid updates!     Tol2 Gateway-compatiable toolbox for studying the nervous system The... Cole lab recently deposited the Tol2 Gateway-Compatible Toolobox, which will allow scientists to quickly... neuorodgenerative diseases. The Tol2 Gateway-Compatible Toolbox is based on the original Tol2kit generated...vectors that have standard cloning sites or gateway compatible cloning sites for easy cloning of your genes ...adds new neuronal tools to the expanding gateway compatible vectors currently shared amongst the zebrafish...

...with increasingly complex experimental conditions. The ability to construct more complex, multi-fragment...fragments in a single “one-pot” reaction to form complex, multi-insert modular assemblies that enable biosynthetic...as enzyme efficiency, stability, and buffer compatibility have placed practical limits on single- or two-step...enzyme cutting results in 5’ four base overhangs complementary to the overhangs of the assembled inserts. After...assembly Intermediate 5- or 12-fragment assembly More complex 24-fragment assembly Single insert cloning efficiencies...Sequencing of blue colonies showed the expected complete sequence for the lacI/lacZ genes (1), while sequencing...setting up assembly reactions in which a single component was purposefully omitted. Since any assembly is...

... Nextflow script. With our completed pipeline, we could adjust the compute resources when needed, for ... number of deposited plasmids increased, the accompanying sequencing data collection and analysis eventually...needed to transform the raw sequencing data into a complete plasmid sequence that would be ready for our QC...data pipeline is to perform several sequential computational tasks for which existing script(s) have been...containers are they are designed to ‘contain,’ in a complete package, all of the pieces of software that depend...is sequestered from other programs running on a computer, so it safely operates in isolation, and can be... be easily shared with others without any complicated setup. We created, tested, and debugged each of ...

...before vitrification, they can be completely denaturated (left) or complexes may dissociate  (right). B) Protein...determine structures at comparable or higher resolution than using more complex protective methods or detergents...evolved optimizations for prime editing CHARM: A compact epigenetic silencer Recombinant antibodies for ...for low-abundance targets or natively isolated complexes. To address these limitations, the Funabiki Lab...linker histone H1.8 in Xenopus nucleosomes and in complex with its chaperone NPM2, but the technique should...tardigrade LEA, RvLEAMshort, protected samples and was compatible with the buffer additive MgCl2 and the crosslinking...other viral delivery methods, allowing all the PE components to be delivered in a single package. In a recent...

...tasks that can be accomplished quickly and things that will take longer. Completing the quick and useful...project they’re a part of, completing them will give me some sense of accomplishment. I will prioritize which...its utility) How much time it will take you to complete the task When picking which tasks to actually...will swiftly give you the wonderful feeling of accomplishment while the longer tasks will give you larger...larger payoffs later. Don’t be afraid to accomplish some quick but moderately useful tasks at the expense ...the long and highly useful tasks. The quick accomplishments will likely energize you. For example, let'...experiment. The first way, it will take you 3 days to complete the entire experiment, but you’ll have to spend...

...user to compare the emission spectra between all of the markers in the panel. This complexity index ranges...choice for overlapping markers. Compensation/reference control(s) Compensation or “reference” controls are...detector for PE. In order to correct for this, compensation has to be applied to “subtract” this spillover...up a small portion of that spectra to use for compensation.     Figure 1: The emission spectra... discern one marker from another. Instead of compensation, which relies on user adjustment, spectral flow...cytometry relies on “unmixing.” Unmixing uses a complex mathematical algorithm and single stained reference...series of histograms flipped onto its side and compressed into a heat map. BV421 peaks in V1, with secondary...

...which can compare up to five editing experiments at a time, and batch analysis, which compares hundreds...edits performed over multiple experiments and comparing the robustness, accuracy, and speed with existing...the accuracy of ICE analysis results was highly comparable (within r2=0.96) to that of NGS data. Advantages... functional KO of the targeted gene.   Analyze Complex Edits   Traditional Sanger sequencing-based analysis...analysis tools are unable to detect or analyze complex edits, such as those generated by multiplexed delivery... How ICE works   Following delivery of CRISPR components into target cells, genomic DNA from both edited...populations is PCR-amplified and Sanger sequenced. ICE compares these sequence traces to give a detailed analysis...

... the “damage” and producing a complete plasmid. Besides being compatible with any vector, the SLIC protocol...overhangs perfectly match up. They just need enough complementary ssDNA to hold the plasmid together, even if ...if there are gaps or flaps remaining. This incomplete plasmid looks like a homologous recombination intermediate...either by annealing a mixture of products or from incomplete PCR — although higher DNA concentrations are ... separate PCR products (middle branch); or by incomplete PCR, which skips the final extension step to ...single-stranded overhangs (right branch). The mixed PCR and incomplete PCR methods yield a minority of usable product...the linearized vector by T4 digestion; then the components are combined and annealed. The resulting recombination...

...tissue and compare the IHC results to both NeuroMab and the Allen Brain Atlas. The comparison with two ...what you’re comparing your sample to. For example, we use our negative control to compare exposure times...cannot be reproduced and projects that cannot be completed. A 2015 review analyzing and explaining the antibody...reliable (wouldn’t perform differently over time), compatible with the desired applications (such as immunohistochemistry...techniques on rodent brains and is expanding to more complex brain systems such as non-human primates and humans...polyclonal antibodies because of their immunoglobin component that can be produced in large “copies.” This homogeneity...monoclonal antibodies; Addgene collaborates with these companies to create and provide general availability of ...

...is mediated via individual neurons. Neurons are composed of a cell body, with dendrites receiving incoming...tracing experiments, brain tissue is injected with compounds that intracellularly spread away from the site...distant brain areas. It is desirable for tracing compounds to outline neurons in the direction of information...proteins facilitate transport of neuronal tracing compounds and this is an area that requires further study... neuronal tracing methods Traditional tracing compounds either produce a colorimetric product or are fluorescent...health. Another limitation of traditional tracing compounds is that their diffusion across the synaptic cleft... These methods are less neurotoxic and better compatible with other neuroscience methods such as electrophysiological...

...more about the company’s program. Once you’ve found a few positions to apply for, compare and contrast ...eventually work at the company at which you’re doing your postdoc? Some companies, such as AstraZeneca,... open up. Other companies, such as Genentech, will not. The point is that each company has a unique approach...people involved is staggering. The process is complicated. If you’re interested in the drug discovery process... publish, but this can vary depending on which company you choose. If publishing is important to you, ...that your project will not involve proprietary compounds or information. Where publication is expected,...meetings. How do I get an industry postdoc? Many companies have industry postdoc positions, including AstraZeneca...

...these components are necessary to efficiently reconstitute a functional viral polymerase complex and utilize...proteins are provided in trans (see Other Important Components of EBOV Minigenome System). However, minigenomes...RNA viruses (reviewed in 2, 3). Other important components of the Zaire ebola virus minigenome system Similar...with the viral polymerase, which is formed by a complex of two proteins: the large (L) catalytic protein...-stranded RNA minigenome. The viral polymerase complex then recognizes the encapsidated minigenome as ... genes in this plasmid would be more difficult compared to if each gene were encoded in separate (and ...particular observation can be easily studied as each component can be examined independently. In addition, experiments...

...antibody can be used. In a competitive ELISA a multiwell plate is coated with a competing antigen (green).  The...antibodies are used.  Competitive ELISAs Scientists typically use a competitive or inhibition ELISA when...converts into a detectable product. Scientists compare the signal intensity of the sample to that of a... of ELISA are direct, indirect, sandwich and competitive. In a direct ELISA a reporter enzyme conjugated...sample, the less antibody available to bind to the competing antigen. In direct, indirect and sandwich ELISA... level of antigen in the sample whereas in a competitive ELISA the reporter signal is inversely proportional...molecules that may bind non-specifically to assay components and increase background staining. Direct ELISAs...

...issues in the past, or how completely different teams are tackling comparable conflicts. Is this level ...cooperation: avoiding, competing, accommodating, collaborating, and compromising. Though there may be a...Skills series, this post is not meant to be a comprehensive guide to Conflict Resolution. There are countless...conflict is actually critical in the business world. Companies and organizations that avoid all conflict can ...with whom you strongly disagree, is a critical component of conflict resolution. Having empathy does not...course many other types of conflict resolution. In compromise, you are trying to come to a consensus that all...parties will accept. If you are able to broker a compromise well, you can help everyone walk away from a ...

...so simple, and engineering complex cellular behaviors requires making complex manipulations to cells. For...assembly. Simply put, it assembles individual DNA components into larger units or circuits with functional...Transcription units (TUs), which are functional units composed of multiple parts transcribed together. They can...and end with a terminator. A simple TU could be comprised of just three parts: promoter, gene, and terminator...MoClo. By a similar principle, TUs can be further complexed into multi-TU constructs, known as multigene constructs...promoters, UTRs, genes, tags, etc.) into MoClo-compatible vectors. Level 1: Assembly of multiple Level ...interact with other proteins and with DNA, and these complexes regulate the transcription of specific genes. ...

... homolog of the yeast TREX-2 mRNA export complex: components and anchoring nucleoporin. Lu et al. Plant...generally known as Bimolecular Fluorescence Complementation (BiFC). It can provide a qualitative or quantitative... or even single-particle tracking of the bound complex. There are so many FPs available, with many ways... In several cases an FP(1-10) fragment may be compatible with multiple FP(11) fragments, even from a different...because they are used with an already-available complementary fragment. Next, let's look at a round-up of...protein fragments for bimolecular fluorescence complementation analysis under physiological conditions. Shyu...

...the key components of Nanoblades, how their editing efficiency and off-target effects compare to existing...the following four components into 293T cells. Figure 1: The key components of Nanoblades. Image... primary cells. Comparing Nanoblades to Existing CRISPR Delivery Methods When compared head-to-head with...for HDR editing and mouse-model generation. Key components of Nanoblades Nanoblades are a Cas9 RNP delivery...Nanoblades also had lower off-target effects when compared to DNA transfection of gRNA and Cas9 expression...of RNPs generally having lower off-target edits compared to DNA transfection.  Homology directed repair...helps form DNA and retroviral-particle or VLPs complexes. The team found that "all-in-one" Nanoblades facilitated...

...Starch-based compostable bags for kit components Recyclable Yes, all kit components and packaging... is completely degraded. Cloning Grade DNA (high quality/low quantity) – You can usually complete a cloning...packaging Yes, most kit components and packaging Yes, most kit components and packaging Parts Sold...can use to test or compare kits before deciding which one to buy. Table 3: Comparison of popular plasmid...a relative term — even armed with the highest-competency cells you will still struggle to get transformants... very similar. However, keep in mind that kit components (i.e. buffers, columns, or beads) are designed...can affect the lysis buffer ratio and result in incomplete lysis. You may be able to scale the amount of...

...at major companies.  The majority of these meetings acted as a platform for significant company advancement...their 20’s who’ve built multibillion dollar tech companies using only laptops and some server space. But... find an extra layer of difficulty launching a company in the health sciences without those critical three... into commercial products and next generation companies.  We launched in the early spring of 2015 and ...Before even beginning the process of launching a company, you need to determine whether or not your idea...your idea, you will rarely succeed in launching a company without partners.  You need a dedicated team that...funding bodies shy away from one man teams.  The composition of the management team is more important than...

...proteins going completely through the membrane or larger proteins not transferring completely onto the membrane... can arise in this step: protein degradation, incomplete lysing/insoluble proteins, and protein aggregation...s membrane-bound or located in a subcellular compartment, you may want to try an SDS-containing detergent...developed for isolating proteins from a specific compartment. If your proteins are nuclear- or DNA-binding...visible to the naked eye; a fluorescent ladder compatible with your imaging system; or a combination of...protein of interest is over-transferring (going completely through your membrane to the other side), you...small and medium proteins. If your proteins are compatible with electroblots/dry transfer systems, and you...

...QCascade complex surveys the genome and binds its target DNA sequence. The transposase complex binds the...around in the lab.  Let’s take a look at the key components of transposons, how they are classified, and ... in both prokaryotic and eukaryotic genomes.  Components of a transposon system Transposon: The transposon...is left behind. When the transposon-transposase complex finds its target site, it integrates, which produces...TEs You can think of autonomous transposons as “complete transposons” since they encode the protein they...transposase, SB100X, is ~100-fold more efficient compared to first generation SB transposase (Mátés et al...

...homology arms (60-70 bp) is added to the complex. This complete transfection mix, i.e. csRNP plus ssODN... Cas9 RNP complexed with 2-part cr:tracrRNA tags genes more effectively than Cas9 complexed with sgRNA...immunoprecipitated - along with its associated protein complexes - from the cell-lysate using antibodies against...against the target protein. The captured protein complexes are then analysed by mass spectrometry to identify...recombinant Cas9 protein to form the ribonucleoprotein complex (csRNP, Figure 1). We use in-house purified Cas9... cost-effective and highly active. After csRNP complex formation, a single-stranded donor DNA (ssODN) ...experiment depends largely on how efficiently these components are delivered into cells; for each cell type,...

...sometimes you want a tool that is a little more compact — more of an antibody fragment rather than the ...Consider the classic IgG antibody (Figure 1): it is composed of four peptide chains (two identical heavy chains...The Fc region is the “tail” of the antibody and comprises portions of the constant regions of the two heavy...on the other hand, are the “arms” of the Y and comprise the entire light chain and a variable and constant...portion. For example, Fab fragments are molecules composed of just the Fab portion of the antibody. Fab fragments...fragments. Size: ~50 kDa. Fv - These fragments are composed of just the variable domains of the heavy and ...Variable (scFv). Size: ~25 kDa.   Figure 2: Comparison between a full IgG antibody and common antigen...

... These are sandwich, competitive, direct, and indirect. Sandwich and competitive ELISA use antibodies ...et al., 2019.   Competitive ELISA Similar to a sandwich ELISA, the competitive ELISA is also used ... will bind to the competing antigen in the wells while the protein-antigen complexes will be washed away... be adapted to a competitive format. Figure 2: Overview of using Competitive ELISA to quantitatively...epitopes on the target protein, so they do not compete with each other. This antibody is also conjugated...protein of interest in the original sample. The competitive ELISA is best used for detecting antigens that...sandwich ELISA. While the most common type of competitive ELISA is a modification of the sandwich ELISA...

...structurally resemble any other known proteins; they are completely unique to the CRISPR system (Nishimasu et al....conformation.   Fun Fact! Even though the Cas9:RNA complex is well described in the closed conformation, recent...electron microscopy studies have also found Cas9:RNA complexes in the open conformation (Cofsky et al., 2022)...guide RNA, prearranged for base pairing with a complementary DNA strand. These 10 nucleotides are the “seed...pyogenes Cas9 in the apo state (PDB ID 4CMP, top) compared to the guide RNA-bound state (PDB ID 4ZT0, bottom...the PAM sequence and doesn’t mind if the PAM complementary strand is mismatched.   Figure 5: S. ... found a PAM site, Cas9 needs to check for complementarity with its guide RNA. But how to test DNA bases...

...endogenously expressed SCF complex members, an E3 ubiquitin ligase complex. Finally, when auxin is added...can offer over other protein control methods and compare the commonly available types of degron tags. What...removed. The degradation and reversal are rapid compared to other systems because the system modulates ...other systems reviewed here. The only required components are 1) a SMASh-tagged protein and 2) asunaprevir...fusion protein with the endogenous CRBN E3 ligase complex which will polyubiquitinate the fusion protein....target protein-of-interest and the CRBN E3 ligase complex leading to ubiquitination and proteasomal degradation...reviewed here. The AID system relies on three components. First, the protein of interest must be AID-tagged...

...a single enzyme. Compared with previous research, which required protein compartments to co-localize multiple... antibodies for aminoacyl tRNA synthetases Nanocompartments for yeast bioproduction pathways The CRISPR...Research Center developed a pooled plasmid library encompassing all possible amino acid mutations of the receptor...many aaRSs exist within a multi-tRNA synthetase complex, the team investigated whether the generated antibodies...antibodies were able to immunoprecipitate the entire complex in three different cell lines. The Gräslund lab...Clouse Claudia Vickers’ lab has created a novel nanocompartment for transporting proteins in yeast by using... anchors to VP1, which self-assembles into a compartment encapsulating the protein of interest (Figure...

...experiment requires delivery of, at minimum, two components: the Cas9 protein and a guide RNA (gRNA) targeting...valuable to scientists, there are some potential complications that must be considered when using this method...alternative approach, which avoids many of these complications, is to directly deliver a ribonucleoprotein ...ribonucleoprotein (RNP), consisting of the Cas9 protein in complex with a targeting gRNA, to your cells of interest. Cas9...cleaving genomic targets with similar efficiency as compared to plasmid-based expression of Cas9/gRNA and can...CRISPR experiment is the generation of the RNP complex.  While one option is to purchase the Cas9 protein... by commercial vendors such as IDT. These two components are then incubated together to form the RNP. ...

...the viral components are small enough to enter the nucleus through the nuclear pore complex. This feature... methods. Across these methods, the following components are needed: γ-Retroviral transfer plasmid encoding...similar to lentiviral packaging methods. The three components described above (envelope, packaging, and transfer...slightly different isoforms of these packaging components. Therefore, lentiviruses may not be efficiently...interchangeably. Are γ-retroviruses replication incompetent? Yes, as with lentiviruses, the genes required...plasmid backbone is meant to be descriptive of the components found within the backbone. For example: pLXSN...particle containing Matrix, Capsid, and Nucleocapsid components. Pol Precursor protein containing Reverse...

.... They are made up of compensation beads and a conjugated antibody. Compensation beads are a mixture between... single color control prior to compensation (B) and after compensation (C) with APC-Cy7.   FMOs FMO...your experiment, and they are non-specific. The compensation beads can bind to any primary antibody you add...up your voltages and can also be helpful for compensation. You should have a single color control for ...They may also be used as a starting point for compensation (Fig. 1B-C). However, it’s important to note...and not experimental samples, the voltages and compensation you set from your single color controls may ... the reference plots provided by the antibody companies or check the literature. Pro tip! Early gates,...

...Delivering CRISPR components to plant cells Once you have selected the correct CRISPR components for your application...deliver these components to your target cells. Remember, efficient delivery of CRISPR components is essential...organisms, some plant-specific changes to CRISPR components are necessary to enable genome editing in plant... academic researchers through Addgene. CRISPR components for plant genome engineering CRISPR can be used...genome of plant cells. The structure of the gRNA (composed of a ~20 nucleotide targeting sequence and ~75...line will result in a failed experiment. CRISPR components can be expressed stably or transiently depending...delivery method and cell type in question. CRISPR components can be delivered and expressed transiently using...

... Note: hypoxanthine (shown) is the nucleobase component of the nucleoside inosine. Created with BioRender.com...and one-ring pyrimidine base. This is a far more complex chemical task, not a job for a single enzyme. ...bind DNA, Cas9 requires (1) a guide RNA, (2) a complementary DNA sequence, and (3) a protospacer-adjacent...removed the uracil glycosylase inhibitor (UGI) components, which inhibit BER during cytosine-to-uracil ...developed two fusions of a CBE with other BER components: the DNA repair protein rXRCC1, or the DNA binding...separate and preventing repair based on the complementary strand. This could explain why the edited U ...original G is not primarily converted back to a complementary C. Optimized base editing with eOPTI-CGBE and...