...Snapp’s blog post shows. To avoid these issues, Subach et al. and Tsuboi et al. developed monomeric FP...mK-GO from Tsuboi et al. and the FT series from Subach et al.) were derived from previously developed ... while attempting to enhance mKO in other ways, Subach et al made a concerted effort to develop their ...see Table 1 for emission and absorption spectra). Subach et al’s directed mutagenesis also produced the ...Monomeric FP Timers and Associated Plasmids from Subach et al. Protein Excitation (nm) Emission (nm)...following its subcellular localization overtime (Subach et al.) Monitor protein expression dynamics in ... (2001): 29621-29624. PubMed PMID: 11408473. 4. Subach, Fedor V., et al. "Monomeric fluorescent timers...

...application cycle becomes much more complex. “How do I reach out to potential mentors?” “Where do I start with...research that aligns with your interests and goals.  Reaching Out to Potential PIs Even if you’re applying to...application process. Many institutions recommend that you reach out to potential advisors before submitting your...your application that you intend to work with, so reach out to three in case one isn’t able to respond to...your email. During my PhD application cycle, I reached out to around twelve professors and heard back ...you determine if the program is the right fit. Reaching out to PIs also means doing plenty of mini-interviews...applications can get to know faculty, students, and each other. Before COVID-19, financial and logistical...

...hierarchical approaches using different Type IIS restriction enzyme specificities at each step. Factors...Our work demonstrates that it is now possible to achieve 20+ fragment assemblies with both robust efficiency...Additional confirmation of accurate assemblies was achieved by the sequencing of plasmids isolated from blue...illustrates how the volume of the 1 ml outgrowth spread on each transformation plate can be manipulated to result...fragment lacI/lacZ cassette assembly was easily achievable with high levels of transformants and low backgrounds...researchers continue to test the limits of DNA assembly approaches with increasingly complex experimental conditions...

...specific UV wavelengths or chemicals (Polstein and Gersbach, 2015; Zetsche et al., 2015). These types of CRISPR...native conditions, the less activation you can achieve using a CRISPRa. Your gene of interest might already...your system is working as expected. Finally, for each gene you want to activate, you should also be ready...Dow D, Buser-Doepner C, Bertotti A, Trusolino L, Stronach EA, Saez-Rodriguez J, Yusa K, Garnett MJ (2019...engineering parameters via a library-on-library approach. Nat Methods 12:823–826 . https://doi.org/10.1038...JL, Davidsohn N, Housden BE, Perrimon N, Weiss R, Aach J, Collins JJ, Church GM (2015) Highly efficient...doi.org/10.1016/j.cels.2018.04.011 Polstein LR, Gersbach CA (2015) A light-inducible CRISPR-Cas9 system...

...colors to label an organ or entire organism. Because each cell can have multiple copies of the transgene that... colors when a combination of XFP are expressed. Each cell remains the same color for its entire lifetime... as many as 16 copies of the injected transgene, each of which could recombine to activate the expression...expression of a different fluorescent protein. Therefore, each cell could express a unique combination of all of...construct, dBrainbow includes small epitopes fused to each XFP. Secondary antibodies that target these epitopes...onto a tamoxifen-inducible ubiquitin background, each cell in the mouse that expresses Cre undergoes exactly... exactly one recombination event, leading to each copy of the transgene expressing either YFP or CFP, ...

...antibodies specific to each fluorophore. Third, Brainbow-1 and Brainbow-2 each contained a “default” state...construct contains two tandem invertible segments, each with two fluorescent protein coding sequences in...then successfully generated custom antibodies to each of these proteins and confirmed that there was no...opening the door for immunostaining analysis. To achieve even cell labeling, they generated farnesylated...-EF1a-BbTagBY. These constructs contain two XFPs each due to size limitations associated with AAV; co-... Nov 1;450(7166):56-62. PubMed. A technicolour approach to the connectome. Lichtman JW, Livet J, Sanes...

...substitutions (or deletions) in a tailored manner. The approach can be used in conventional cloning (to introduce...reverse primers are designed to be complementary, but each primer may extend beyond the complementary region...larger band (not shown). As a footnote; a similar approach can be used to identify Cas9/CRISPR-induced genome...template for the mutagenesis reaction. Alternative approaches to site directed mutagenesis Point-mutagenesis...PCR-introduced mutations can make alternative approaches more favorable if you want to introduce a point... introduce a frame-shift mutation. This can be achieved using a restriction enzyme that recognizes a unique...directed mutagenesis is one of the most popular approaches for introducing minor modifications to a plasmid...

...gills. It attaches there until they mature and changes sex from male to female. Then, it detaches from the...their foraging trail. They climb upwards until they reach a point where the temperature and humidity favors...Vampirovibrio chlorellavorus is a predatory bacteria that attaches to the cell wall of green algae (Soo et al., 2015...eyestalks. Once ingested by the snail, the larvae reach the eyestalk and transform them into a pulsating... grasshopper harbouring hairworm: a proteomics approach." Proceedings of the Royal Society B: Biological...

... MS, McKenzie A 3rd, Demarini DJ, Shah NG, Wach A, Brachat A, Philippsen P, Pringle JR. Yeast. 1998 Jul... in epitope tags? Others may be interested in attaching epitope tags to their genes of interest, allowing...complete collection of yeast tagging modules with each possible combination of protease site (TEV or PreScission...and selection marker (KanMX, HphMX, or His3MX).  Each PCR product from this collection will yield an insert...Longtine MS, Shah NG, McKenzie A 3rd, Steever AB, Wach A, Philippsen P, Pringle JR. Yeast. 1998 Jul;14(...

...could then be used to examine both inter- and intrachromosomal dynamics, and how they change in response ... to SpCas9 and its orthologs NmCas9 and St1Cas9. Each ortholog was fused to a different fluorescent protein... to differences in the PAM sequences required by each ortholog, a gRNA designed for one dCas9 should be...turned their attention to mapping pairs of intrachromosomal loci. The technique successfully resolved ... this work represents the first mapping of intrachromosomal loci, a major benchmark in characterizing ...under lower illumination settings, lowering photobleaching and phototoxicity concerns and extending the...

... the most approachable for LEGOs. Stop motion consists of taking many pictures, one for each position ...video. We’ve grown attached to the work, and I confess that, when I finish building each scenario, I go “...one doing experiments in France. We can learn with each other through globalized, collaborative thinking...help further unlock the potential contained within each. There must be some way to use LEGOs, which are ...

...gRNA targeting approach is then used to direct the complex to a specific locus. This approach provides a ...a technique that utilizes phage recombination machinery to promote homologous recombination of linear ...resistance E. coli carrying the phage recombineering machinery are first electroporated with pCas9. Then, pCRISPR...repair template is supplied as a dsDNA fragment. Each round of editing takes two days, and the pTargetF...method in E. coli, Jesse Zalatan’s lab used an approach to identify known proteins involved in the activation...polymorphisms, including antibiotic resistance genes. This approach successfully targeted specific organisms when ...et al., 2018).  Base Editing Among the newest approaches that leverage the remarkable properties of CRISPR-Cas...

...light microscopy-based neuronal reconstruction. To achieve stochastic multicolor labeling, Brainbow used the... Stochastic multicolor labeling is possible when each of the XFP genes is introduced at ~2 copies per ...you use AAVs, it typically takes a few weeks to achieve optimal expression of XFPs. Any longer and the ... introduced at ~2 copies per cell per color to achieve the highest color variations. Adjust the plasmid...Tetbow AAVs - You will have to wait a few weeks to achieve an adequate expression level of the Tetbow AAVs...critical to express a minimal amount of tTA to achieve highest expression levels. Clearing large tissues...

... target. There are a multitude of options for achieving success in a science policy career with many opportunities...background, learning the science part on the job. Each path to science policy is unique and this creates...that you can attend to help build relationships.  Reach out to a science policy professional over Twitter...s talking about themselves--so don’t be shy and reach out! Someone from your alumni community is a great...audiences during your time in graduate school through outreach organizations and science blogs. Other avenues...organizations, working in student government, and teaching and mentoring others (see more in Figure 2). Starting...resource guide including conferences, societies, and outreach groups. Grab a virtual coffee to chat with someone...

...complicated (and again, expensive) the different machines are, and how many regulations are in place to ...proteins. FD-CF reactions are also cost-effective, as each one is as cheap as 10 cents, making activities designed...in a variety of settings. If you are a biology teacher at the elementary, middle, or high school level...who can share their experiences and knowledge with each other to help make learning biology more fun – bit...developing synthetic biology based tools for science outreach and advocacy. You can email her at [email protected]... on the Addgene Blog Find planarian tools for teaching developmental biology Use the pathways over time...

...Since there is a limited amount of surface area on each cell, the way to get more AAV in contact with the...proportion of AAV particles that make it through each part of the cell on their way to the nucleus where... as many viral particles must infect a cell to achieve visible expression of the protein. However, expression...expression of receptors may not need to be as high to achieve an observable signal in your experiment, therefore...looking for effective gene expression. How do you achieve high titers in the first place? Some AAVs are known...solution. Add a bit less buffer than you would to achieve the exact desired concentration to avoid over-diluting...

...than KillerRed, with KillerRed reaching greater than 75% photobleaching after just 1 minute of exposure...produce ROS - targeted cell ablation via ROS is achieved through localized expression of the FAP. A new...portion of the complex and is derived from the malachite green (MG) dye, but has been modified with the...longer exposure times (5 minutes vs 10 seconds) to achieve the same level of killing as Mg-2I-dL5**. Mg-2I-dL5...exposure. MG-2I-dL5** experienced minor bleaching after longer exposure times. Additionally, KillerRed requires...

...love, and even some you’ve likely never heard of. Each entry displays the following information for a Cas... get you started, we’ll give a quick overview of each CasPEDIA feature. Activity features: CasID CasPEDIA... will display both the cis and trans activity of each entry. For example, SpyCas9a (one of the most frequently...host factors required for CRISPR processing. For each enzyme, it will also show you how multiplexed arrays...site. In the Experimental Considerations section on each enzyme page, you'll find a link to search Addgene's... as research progresses, but you are invited to reach out to suggest new entries or even new features ...

...non-interacting with) the cells’ own natural translation machinery; these are often incorporated into the cells via...existing GCE tools are not very robust. When using GCE, each component of the experiment system (including media...temperature) may need to be individually optimized for each of the three factors: the ncAA, the tRNA, and the...trade-offs. This balance can be extremely tricky to find: each of the factors can negatively impact both translation...scientist interested in, or currently using, GCE to reach out to them and let them know directly what protocols...  Rachel Leeson...

...associations: common colds, COVID-19, norovirus (the stomach flu), and many more. Their infectious nature allows... viral particles, the functional infection unit. Each particle has a viral envelope, which fuses with ...They can also be directly injected into hard-to-reach organs, such as the brain. Secondly, some viruses... post. If a viral vector seems like the right approach for your experiment, you’ll need to decide exactly...adenovirus, and adeno-associated virus (AAV). While each virus allows you to infect cells and deliver cargo...and retro viruses do. They are generally used to achieve transient, high expression of the relevant cargo...