...Athanasios Lampropoulos et al. "GreenGate - A Novel, Versatile and Efficient Cloning System for Plant Transgenesis...

...Plasmid 1: B. subtilis knock out and knock down libraries. Listen to Our segment on the B. subtilis libraries...you'll learn about new libraries for studying B. subtilis biology, plasmids for enhancing iPSC production... b. subtilis...

...recently deposited 2 Bacillus subtilis libraries with Addgene – the B. subtilis Single Gene Deletion Library—Kanamycin...target specific neuronal subtypes was not available until now. In order to target specific neuronal subtypes...to light. Robert Campbell’s lab illustrates the utility of PhoCl in a variety of settings. For instance...the podcast Segement on the pPSU Plasmids B. subtilis knockout and knockdown libraries The Gross Lab... Essential gene Knockdown library. Although B. subtilis has a fully sequenced genome, many applications... to advance our collective understanding of B. subtilis gene functions and interactions. The single-gene...single-gene deletion library is derived from the B. subtilis 168 strain and contains knockouts for all 3984 genes...

...break through the resolution barrier while still utilizing fluorescence microscopy, is another option....more accessible imaging equipment, ExM protocols utilize relatively inexpensive reagents and are readily...fluorescent proteins (Gambarotto, et al. 2019; Tillberg, et al. 2016). That being said, the anchoring,... between them to ensure the expanded images are still a good representation of reality. Third, expansion...secondaries, can improve signal strength (Sherry and Stiles, 2022). Fourth, signal continuity can appear reduced.../doi.org/10.1371/journal.pbio.3001020  Chen F, Tillberg PW, Boyden ES (2015) Expansion microscopy. Science...https://doi.org/10.1098/rsta.2021.0110  Sherry DM, Stiles MA (2022) Improved fluorescent signal in expansion...

...have waited. Both tasks have some utility, but I lost all the utility (fun) of eating with friends by splitting... meeting up with friends, but didn’t realize it until it was too late. I didn’t spend enough time thinking... two main things: How useful the task is (its utility) How much time it will take you to complete the...should err on the side of things that have the most utility; i.e. those things that will be most useful for...option. Even if the full experiment fails, you’ll still have made small accomplishments each day that should...and think about what they mean in terms of the utility of your tasks. In my cell splitting anecdote, I...hood may have prevented me from splitting cells until late in the day anway. There are a few things I’...

...within an E. coli cell, but vectors can be widely utilized across many different cell types and each one ...can post-translationally modify proteins yet they still share many of the same technical advantages that..., a well-defined genetic system, and a highly versatile DNA transformation system.Unlike most other microorganisms...of these vectors. S. pombe plasmids oftentimes utilize an ARS to aid in high transformation efficiency...markers for yeast Historically, scientists have utilized auxotrophic selection rather than antibiotic selection...auxotrophic strain/ selection element pairs must be utilized or a new combination needs to be created in advance...improvements in antibiotic selection have made utilizing the more traditional drug selection methods feasible...

...FLEx vector design Recombinase-dependent vectors utilize pairs of specific sequences, lox (recognized by...expression, low levels of spurious expression were still observed in many systems. So what is the cause of...of DIO/FLEx vectors, the cause of these events, until recently, was not known. A recent paper from Ed ...post-translational modifications, and other factors that AAV utilizes to gain entry into the cell (Dudek, AM. 2018)....in order to minimize off-target expression while still achieving adequate expression in your cells of interest...recombination rate (0.1% and 0.13% respectively) still required a 200-300 fold dilution to minimize off-target...accuracy and reproducibility of the data and can instill greater confidence when interpreting the outcome...

...joining the conversation on Reddit, but it wasn’t until one of our blog writers posted his article (Developing...00 am and Joanne didn’t have to start answering until 5 pm). The ability to see the questions first makes...that I didn’t have the direct link to send people until the day of the AMA. This made it a little difficult...alter my intro to reduce the number of potentially hostile posters (this advice included leaving out the term...career path development--but the science trainees still have to lead on their own development. Please see...early in the day. Although I didn’t start answering until 5pm, frankly, I was typing far before five. Fortunately...your own professional development etc. database. Still have questions? I am easy to reach and happy to ...

... engineering. When compared to that collective utility, it’s easy to fault the lack of features in a single... can be used to aid design. These databases are still young and will require a collaborative effort from...arbitrary PAM definitions. These newer tools may still prove somewhat useful for the latest NTT targeting...protein, Cpf1. Separately, on-target efficacy is still a concept that only a few tools are trying to solve... workbenches such as MacVector or Benchling. Utility functions: These functions help speed up the sgRNA...single-method design”. Trends: The most common utility functions are batch design or multiplex features...use. The R based package CRISPRseek offers great utility and coupled with Bioconductor should be the first...

...the mandate has been removed, institutions should still go ahead with raises, as for years there have been...planning to raise salaries prior to the injunction are still honoring these plans. We are now collecting data...following the injunction, 59% of all postdocs are still expected to receive salary raises - down from the...to get a salary increase prior to the injunction still won’t after the injunction (see below). Of the ...salaries, the only postdocs we knew for certain to still be getting their raise were: postdocs at institutions...University of Louisville in Kentucky, the raises are still going ahead as planned. As we are receiving data...

...from one cell to another). Bacteria need special fertility genes to be capable of conjugation, and these ...penicillin resistance plasmid can grow in spite of a hostile Penicillium fungus trying to kill them off. While...possess plasmid-borne toxins like colicins and subtilisins to kill other bacteria in the area, freeing up...systems that allow tuning of metabolic pathways. Fertility (F) plasmids: microbial sharing F plasmids contain...shifted over the years, and new classifications are still being created as whole-genome sequencing becomes...Gateway cloning relies on genes found on an E. coli fertility plasmid that also encodes a toxin, CcdB, and the...

...The versatility of CRISPR allows you to play with DNA in a number of ways, from small edits that change...seemingly ubiquitous in nature, they are largely underutilized in research settings — mostly a consequence ...Cas3 — a tactic some CRISPR transposase systems utilize with type I-F Cascades. Figure 2: Cascade...plasmids — such as the Tomoji Mashimo lab and the Tilmann Weber lab — to increase efficiency of these systems...systems, though consistent expression can still be a challenge. A multitasking Cas Most Cas effectors have... potential. Hopefully more people will want to utilize Cas3’s degradative power in the future! References...

...introduced into the B. subtilis genome. Mobile-CRISPRi is then transferred from B. subtilis to the bacterium...CRISPRi into a single bacterial strain, such as a B. subtilis, or a narrow range of bacterial species, such ...integrative and conjugative elements in Bacillus subtilis (ICEBs1) system. ICEBs1 carries the genes needed...bi-parental mating. Following excision from the B. subtilis genome, the Mobile-CRISPRi ICE element forms a...integrative and conjugative element in Bacillus subtilis." Plasmid 86 (2016): 14-25. PubMed PMID: 27381852...

...c-terminal nuclease domain. Feng Zhang's group utilized the bi-lobed architecture of Cas9 to engineer ...activation Optogenetics is a powerful tool that utilizes light to control and monitor individual living...CRISPR-Cas9 system to create a tool that is quick, versatile, and robust. Dubbed the LACE system for light-...signalling pathways. These LIDs are generalizable, versatile and powerful tools which allow you to easily and... both live cells and whole animals. The system utilizes the bulldozer-like quality of the ribosome during...the hypothesis that most mRNAs are not translated until they are exported to the cytoplasm. Furthermore,...sgRNAs with Cas9 for genome engineering. The versatility and ease of generating complex combinations of...

...refers to as “PAM deserts” in the genome. More versatile and precise than base editing (in certain circumstances...application, and the efficacy varies widely — it still almost always results in higher ratios of desired...Prime editing tools Prime editing is precise and versatile, but it requires careful optimization to achieve...cost of improved efficiency, although both enzymes still approach or exceed PEmax editing efficiencies for...obtain PEs small enough to deliver with AAVs but still efficient at long and complex edits. The team also...this tail is degraded, the truncated pegRNA can still guide Cas9 and compete with full pegRNAs for access...engineered virus-like particles. However, there’s still a long way to go before we see prime editors in ...

...saving the researcher both time and resources (3). Still, researchers generating transgenic animals may choose...helped researchers at a time when sequencing was still in the early days of development. Today, researchers...restriction enzymes are certainly not dead yet! Labs still keep restriction enzymes on hand for cloning and...sequencing (more on that below), restriction mapping may still be used to screen multiple colonies before selecting...expensive sequencing fuels a sequencing revolution that still prevails. Sequencing whole genomes or profiling ...techniques have largely replaced these, these techniques still have useful applications and have informed many ...

...study their function in health and disease, but, until recently, it just wasn’t that easy. In the 1980s...was fascinated by CRISPR and rushed to test it. I still remember the day, March 16, 2014, that I got my ...into either the pronucleus or the cytoplasm of fertilized mouse eggs. Or, to avoid handling embryos ex ...that you can inject the reagents directly into fertilized mouse eggs, circumventing the need for enrichment...transmission of the transgene, this technique is still in need of improvement. Also when working with CRISPR... discovered in an obscure bacterium and that we still have much to learn from biology in all its forms...

...fluorescent sensors. Retroviral vector background Until the development of viral vectors, gene transfer ...membrane pores) transfection. These techniques are still used and efficiently transfer plasmid DNA efficiently...vector. Lentiviral vectors Although retroviruses are still in use, considerable efforts have been devoted to...However, in this vector production system, there is still a potential risk for the generation of replication...however improving the safety of these vectors is still an active area of research due to the possibility...Replication-incompetent lentiviruses with human tropism can still infect the researcher and insert into her/his genome...

... the University of Manitoba. Our lab has been utilizing chemogenetics (Designer Receptors Exclusively ...themselves are not pathogenic, precautions should still be taken when using them. There are suspected cases... useful for a wide variety of experiments, care still needs to be taken when selecting which particular... other viruses, multiple freeze thaw cycles can still reduce AAV titer. For long term storage, make > ... vs 7 days vs 14 days etc. To decrease the time until expression, it is possible to use AAV constructs...Additional Resources on the Addgene Blog AAV: A Versatile Viral Tool for Gene Expression in Mammals A Match...

... NpHR, can even be expressed simultaneously and still be individually activated with different lights ...assays can be done on zebrafish at 5 days post fertilization, including escape behavior and spontaneous swimming...swimming. When startled by acoustic, visual or tactile stimuli, larval zebrafish react by performing a...immobilized in agar at the head with their tails still free, and different regions of the spinal cord were... offered by the optogenetics toolbox and the versatility of the zebrafish model organism, the Isacoff ...