...multi-protein complex, stabilized promoters can eliminate variability between the different components. When ... origin of replication will be overrepresented compared to those further away. As a result, variable gene...located near the origin will be over-represented compared to those further away. While constitutive promoters...gene expression during shifts in growth rate as compared to a constitutive promoter. To simulate the increase...

... changes when it goes from a more neutral pH compartment, ­­like the cytosol, to the higher pH of the ... to other pathways, it is standard practice to compare degradation rates between samples treated with ...the brightly-coloured Australian parrot. It’s comprised of two tandem fluorescent proteins: a relatively...Rosella’s excitation and emission spectra. Component Excitation Emission pH Range DsRed.T3 488, ...autophagy markers. It’s particularly useful for comparing bulk autophagy vs. mitophagy, the targeted autophagy...

...CasMINI plasmids Efficient CRISPR editing with a hypercompact Cas12f1 and engineered guide RNAs Yong-Sam Kim...result is a highly efficient Cas system with very compact genome editors and a gRNA down-sized by almost ...activity of Cas12f1 with these modified gRNAs is comparable to that of SpCas9. The group also confirmed that...Xue’s lab also uses paired pegRNAs targeting complementary DNA strands to program large genomic deletions...enzyme with a fully active Cas9 enzyme. PE-Cas9 comprises an active Cas9 fused to the reverse transcriptase...

...available in rAAVs!   Figure 1: Diagram of AAV components      Cargo capacity Empty rAAV has a cargo...conventional single-stranded AAV (ssAAV) and a self-complementary AAV (scAAV) vector. Full-length ssAAV vector...2.5Kb) and you need expression in a hurry, self-complementary AAV (scAAV) may be an option (McCarty et al....take the next step – selecting your AAV and AAV components (serotype and promoter) for use in your experiment... E. Monahan, and Richard J. Samulski. "Self-complementary recombinant adeno-associated virus (scAAV) vectors...

...below) Because off-target sequences have less complementarity to the gRNA, they have a higher propensity ...SpCas9-HF1 generated fewer off-target cuts when compared to WT SpCas9 across a variety of genomic sites...M694A/Q695A/H698A). This new variant displays comparable on-target activity and similar or decreased off-target...off-target activity across the sites tested when compared to eSpCas9(1.1) and SpCas9-HF1. The HypaCas9 plasmids... Hiroshi, et al. "Crystal structure of Cas9 in complex with guide RNA and target DNA." Cell 156.5 (2014...

... The destination vector contains sites with complementary overhangs that direct assembly of the final ...recognition site and permits DNA fragments with compatible overhangs to be efficiently assembled. Scientists...inverse orientation. This allows for multiple DNA components (promoters, genes, terminators, etc) to be assembled... reaction. For detailed Golden Gate protocols, complete with helpful tips and tricks, see The Sainsbury...system is an incredibly powerful tool for cloning complicated constructs in a single, high-efficiency step....

... don't do anything. Viruses are complicated and they perform complex biological tasks, both of which ...undergo DNA rearrangements. Depending on the composition of the viral vector and the repeat sequences ...perform a diagnostic restriction enzyme digest and compare it to some reliable controls. 2. Viral stocks may...ensuring viral production is to perform PCR against components of the viral vector using the harvested viral...

...benefits and drawbacks of different methods (for comprehensive protocols for all of the methods discussed here...quantified using qPCR primers targeting specific viral components such as LTRs, gag, WPRE, antibiotic resistance-genes...genomic DNA isolated, and primers targetting viral components or the transgene itself are used to quantify ...critical that standard conditions be used when comparing different batches of virus. In addition, some ...PMID: 19300443. 2. Geraerts, Martine, et al. "Comparison of lentiviral vector titration methods."BMC biotechnology...

...Revolution Bioengineering.  Last year we started a company. Revolution Bioengineering is two responsible adults...attend conferences makes it very challenging to compete in today’s tough academic or industry job market... this technology and angry at the agriculture companies that produce GMO food plants. When a press piece... – although we didn’t figure out how we would accomplish the color change until a few months later. RevBio... of our business, because we are building this company, and that’s what we want it to do. It’s exhilarating...

...expression baseline. Copy number is then determined by comparing results to a standard curve. For CNV assays, it...gives an absolute value of DNA concentration. By comparing the number of positive droplets for the gene of... through concentration values, rather than by comparison to a standard curve, and most results can be ...determination to move forward with your experiment?  Complexity of the gene of interest Some gene sequences ... S., Hollox, E. J., & Boon Peng, H. (2015). A comparison of assays for accurate copy number measurement...

...plasmids, they are generally electroporated into electrocompetent cells to ensure the highest possible transformation...use for your transformation(s). The amount of competent cells you need total and per transformation may...liquid culture, all of the bacteria can grow and compete in the same container, making it easier for a smaller...be its own colony, and will not be in direct competition with other colonies containing smaller, recombined...used for libraries that are similar in size and composition as examples. Table 1 provides an overview of ...

... pooled CRISPR library is a complex mixture. Pooled libraries are composed of many different gRNA-containing... and versatility. CRISPR/Cas9 consists of two components: the non-specific endonuclease (Cas9) and a single...any genomic locus, provided the target is unique compared to the rest of the genome and located immediately...distribution is analyzed in the drug-resistant population compared to a non-treated control group. In this scenario...

...multiplex (Thellwell, 2000). See Table 1 for a comparison of features of several commonly used chemistries...have pipetting errors on the edges of the plate. Compare the CT values within each set of triplicates to...reaction, each target in the reaction should be compared to an individually run sample when developing ...value representing the fold change of each sample compared to your control. But as we said previously, just... See Table 2, (adapted from Wong, 2018) for a comparison of different analysis methods.   Method ...defined log-linear phase   Table 2: A comparison of different qPCR analysis methods. Table adapted...

...one such technique and has been used to produce complex fluorescent reporter constructs. These fluorescent...gene-expression sequences, and more. By combining these components, one can generate constructs to visualize cell...terminus or the 3’ terminus of the ligation product. Comparison to other high throughput cloning strategies There...modules, because neighboring fragments require compatible cohesive ends Engler et al., 2008 Gibson Cloning...facilitate the construction of plasmids with many components. The creators of the MXS-chaining method have...

... takes 1-1.5 hours to complete, while this syringe-based method can be completed in 20 to 30 minutes. ...PCR buffer, where DNA or RNA can be amplified. Compared to a commercial magnetic bead-based kit, this ...this did not reduce cloning efficiencies.  When compared to fresh columns, regenerated columns had similar... reduce the number of spin columns you need to complete your research. Do you know of other nucleic acid...

...feel comfortable speaking on topics that would compliment the STEM curriculum. As it happened, I’d met ...simplistic and I would insult the students. Too complicated and I would quickly lose their interest. I won...careful to keep it relatively simple. I honed the complexity of the talk by running it by the teachers and... For CRISPR, I focused on the fact that, like computers and the internet before it, genome engineering...posts Resources on Addgene.org Check out our comprehensive CRISPR guide Find CRISPR plasmids and resources...

...you’ll want to ensure that your buffer components are compatible with your conjugate and conjugation chemistry...the assay. Where possible, it can be useful to compare recombinant antibodies to their parental hybridomas...majority of antibodies are still equally effective compared to those aliquots frozen immediately post-harvest...Chen E, Haidar JN, Srivastava A, Goldstein J. Comparison of predicted extinction coefficients of monoclonal...

...will link biomolecules to the gel and add gel components to polymerize. Step 3: digest the sample to allow...labels you could use. But many ExM protocols are compatible with conventional antibodies, chemical dyes, ...and least flexible one. Tissue expansion is not compatible with in vivo applications and there’s just not...isotropic expansion depends on how well the gel components penetrate the sample, how well the gel polymerizes... valuable tool, especially when paired with complementary experimental approaches. Since it arrived on...

...collaborations, and commitments to and proof of compliance with the grantor’s requirements as well as state...reports are submitted and project evaluations are completed. To enter and advance the profession, you can ...Professional Development Strategy to increase your competencies and skills in the grants profession. Your professional... and the candidate’s knowledge of the 8 core competencies through a multiple choice exam.  The bottom ...grant professionals and to build your skills and competencies. Becoming a grant professional is rewarding ...

...into a translated protein. As depicted in the accompanying cartoon, they have a multitude of uses including...TAP) once referred specifically to a combo tag comprised of a calmodulin binding peptide (CBP), a TEV cleavage...ProtA IgG-binding domains. TAP has since come to encompass several other tag combinations, though frequently...and Gly residues   This article is not a comprehensive guide to all tags, but rather a quick overview...Biotechnology Journal 2012 , (7): 620-634. Full Text A complete list of free handbooks for protein purification...

...necessarily mean that you are a future employee of the company. It does, however, offer you an opportunity to ...like to work, get online and take a look at the company or institution’s career page, where you’re likely...openings. A good approach is to do this first for companies at which you’ve already made a personal connection...result in a recruiter giving you a call. Many companies enlist the assistance of recruiters for identifying...development center, if one exists. Get online! Review company and institution career pages. Make yourself visible...

...the ability to cleave the RNA target.  Table 1: Comparison on Common CRISPR Enzymes Name  Enzymatic Domains...Amplified nucleotides are combined with Cas 13a in complex with crRNA targeting the sequence of interest and...multiplexing; expressing 5 gRNAs simultaneously produces comparable gene knockdown to single gRNA expression. Although... cell stress, and RNA size distribution. When comparing LwaCas13a to shRNA, they found that LwaCas13a ... Abudayyeh, Omar O., et al. "C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector...

...fields in order to provide useful solutions to complex challenges. As a cross-disciplinary researcher,...Many of the challenges in science today are too complex for any single discipline to solve them on their... disciplines leading the way, the future of complementing published research with video overviews was ...may never access online journals or even use a computer.  Currently, two of the more popular biology videos...derived from research. The platform uses video to complement traditional print journals and expand our ability...

... research compiled in the ELN. Without such references the record of research is incomplete, making it...commercialization office, the CIO’s office, and research computing. The two guiding principles emerging from the ...the record of research captured in the ELN is incomplete. Thus, to support reproducibility an ELN should...select and deposit individual documents or more complex datasets into the desired repository. They also...

...brainstorm session is to start with something completely unrelated to the issue and consider how it could...unique solutions to the same problem. If your lab/company/team is still working towards a culture of encouraging...just as valuable! In 2019, Addgene implemented a company-wide New Ideas Survey. Anyone could fill out the...of which, when implemented, impacted the entire company. It is important to note that the person who submitted... is an incredibly innovative group. Our entire company benefits from their policy! Perfectionism When ...

...sgRNA targeting the genomic site of interest. We compared the pCas/pTargetF system with the updated pEcCas...system.   Advantages of the pEcCas/pEcgRNA system Compared with the original system, the pEcCas/pEcgRNA is...strategy for plasmid curing, providing users with a complete editing process. The MUCICAT system While the ...dehydrogenase expression that is 2.6 times higher when compared to the industrial strain. In our metabolic engineering... pQCascade) with 4 rounds of transformation to complete the N-acetylglucosamine strain library construction...

...applying to the specific company or lab. Take some time to research the lab or company via their papers and...place to state why you’re applying to the job, company, and/or lab, and how you feel connected to their...apply for [title of the position] at [name of the company/organization]. I am a [adjective that describes... are interested in working for this lab and/or company. Remember to proofread your cover letter, and ...

...in the targeted brain region after stimulation compared to controls without Chr2. c-Fos is a reliable ... to proceed with your behavioral experiments. Comparison of c-fos amongst animals that receive different...Inhibition of neuronal activation is a little more complicated since basal neuronal activity in your brain region...writing. Derek is also a pianist and an aspiring composer. View his blog drsimonsaysscience.org or follow...applying optogenetic tools derived from direct comparative analysis of microbial opsins." Nature methods...

...revolutionised the way we apprehend molecular complexes and is still a very useful tool.    Like a guitar... These issues can be overcome by choosing a complementary pair of fluorophores. To maximize the FRET signal...to target mTurquoise2 to various subcellular compartments pCEP4CyPet-MAMM Cyan Mammalian Expresses mammalian...Clover pGWF1 Cyan & Yellow Bacterial Gateway-compatible vector to express a gene of interest fused between...especially when you investigate unknown molecular complexes. But it is a parameter that you have to keep in...

... of the policy comprised of numerous objections and concerns. Perhaps least compelling of these were those...to individual researchers. Yet accurate and comprehensible organization of data is essential for efficient...numbers of figures such that many have multiple, complex panels. PLOS, itself, has not finalized its definition...collections and interpretation of data, publishing complete data, rather than just a single projection of ...

...such as cancer since mice better recapitulate the complex interactions between cancer cells, therapeutic ...Scientists can make a new line of flies in only 6 weeks compared to months to make a transgenic mouse line. Over...be used to easily screen multiple therapeutic compounds in one experiment. Zebrafish (Danio rerio) Since...embryonic development studies as their embryos are completely transparent. Thus scientists can easily observe...model organisms, the C.elegans genome is also completely sequenced and over 60% of human genes have an...

...tough. Is the content accurate? Are we conveying complex ideas in the easiest way possible? Are the shots...Jennifer. I had finally found my footing with the company. I’d identified the ways to get the most out the...things like lighting, camera angle, and shot composition.  So back to the drawing board.  Cycle 3: A Return... and availability, a traditional solution was completely out of reach for Addgene.  So, onto research!...  Figure 4: The remote directing setup, complete with wireless video receiver, camera control, ...

...sword Microtubules are one of the most important components of the cytoskeleton. They provide cells with ...you’re interested in that other most-important component of the cytoskeleton – actin? Actin dynamics control...manipulating Rho GTPases based on BcLOV4. These single-component systems take advantage of BcLOV4’s light-induced...powerful enough, what about altering the chemical composition of the membrane itself? The Baskin Lab previously...K. H., & Chow, B. Y. (2022). Designing Single-Component Optogenetic Membrane Recruitment Systems: The ...

...for a Faculty Position" eBook Here 1. Have a competitive CV You don't necessarily need a Nature/Cell/Science... research is about and what you think you can accomplish. No matter how many times I say this, candidates...specific questions and experiments. Nobody EVER complains that a seminar was too easy to understand. Make...detail. Finally, state how successfully completing the research will advance the field, create a...yourself to me by email or in person. If you feel compelled to repay me in some way, I encourage you to help...

...size, while SSC values depend on the structural complexity inside the cell or on its surface. Bear in mind...on the cell’s size, morphology, and structural complexity.   Height, Width, and Area Within FSC and...each coming with a different size and structural complexity. After several steps of tissue processing prior... for a single cell gate. However, make sure to compare H and A (or W and A).   Live/Dead Gating Having...cells stain positively for both markers. When comparing two markers simultaneously, we commonly describe...

... a good story (1). From a deep sea shrimp to a compact powerhouse of bioluminescence, NLuc is 100-fold...yield, brightness and improved maturation time compared to similar orange fluorescent proteins. In their...more than 2-fold brighter signal from Antares as compared to Fluc in this model. The construct for Antares...transfer of energy from one fluorescent protein to a compatible fluorescent protein. FRET can be measured by ...fusion protein called CalFlux VNT. CalFlux VNT is composed of Venus fluorescent protein, the Troponin C Ca...

...lots of hustle and bustle, but the lab was very competitive and not very communal. That didn’t suit my collaborative...choosing a training lab, I strongly suggest you complete a work rotation in the lab if at all possible.... of effort, intimidating use of discipline or competence procedures Threats to personal standing – including...atmosphere makes you successful. Collaborative Competitive Papers have multiple authors One author plus...choose a grad school or postdoc lab. Having a comparison can be very helpful in uncovering strengths and...

...programs. The software has two components - a database of pre-compiled register designs and a search function...sites, promoters, genes, terminators, etc) are composed. Fear not – the state machine design space is ...circuits in general would benefit from a more comprehensive characterization of the recombinases at our ...the Lu Lab at MIT. He is broadly interested in biocomputing and biological circuits.     References 1. N...

...General Counsel at Atalanta, an early-stage biotech company that is developing RNAi therapeutics to treat neurodegenerative...law. He said, “you know, the patent lawyer at my company seems to really like what she does.” I thought ...patent counsel and ran a group at two fairly large companies, Genzyme and Biogen. They’re very different jobs...corporate work, and negotiating contracts with other companies. It's one of those "every day is something different...

..., in vitro screens cannot faithfully model the complex spatial, cellular, metabolic, and cytokine microenvironment... cells, an important immune cell type, in more complex and physiological microenvironments. These screens...immunogenic, a necessity for in vivo studies; (3) is compatible with single gene KO and pooled screening approaches...facilitate the isolation of modified cells; and (5) is compatible with the introduction of unique molecular identifiers...