... from viruses that infect people in nature, extra safety measures, like replication incompetent viral ...the stomach flu), and many more. Their infectious nature allows them to easily deliver their “cargo” to ... make you sick…well, you feel it. But the same features that make these familiar viruses such a pain is...biological tools.   Viruses as biological tools In nature, viruses infect cells and organisms (which we refer...of cell the virus will target and fuse with. In nature, this determines which organisms and cells they...Institute. Its genome, however, isn’t enough to ensure the virus can replicate and spread. Instead, viruses...are required. We will be covering these safety measures in a separate, upcoming post. If a viral vector...

...yeast, are temperature sensitive screens. These involve raising or lowering the culture temperature to exacerbate...Selections do require there to be a culture condition that can apply enough pressure to select for variants of ...individually administered and then outcomes are measured. While the entire genome can be knocked out with... have a screen that doesn’t yield any hits. Make sure you plan ahead and consider these questions to save...though. A selection can take the form of varied temperature, nutrient availability, drugs, and much more....with screens. Selections require there to be a culture condition which eliminates manipulations that do...conditions which convey resistance to a drug. You could culture cells in the drug of interest at a lethal dose ...

...repository, but be sure to let us know if you'd like to write about your plasmids in a future blog post. No...under the control of the cI857 temperature-sensitive repressor. The temperature-controlled expression of the...crystallographic structures available in the PDB. Parton DL, Hanson SM, Rodríguez-Laureano L, Albanese SK...articles. These brief articles highlight the main features and applications of a partiular plasmid or set...arduous. The cloning process alone involves much literature searching, DNA synthesis, and plasmid assembly.... The pooled library, designed to be used with cultured Drosophila cells, targets 13,501 fly genes with...will create new traits in bacteria. Wang, et al. Nature. 2009. PubMed PMID: 19633652 Court, et al. Annu...

...and one NHEJ-repaired indel, often occur. Figure 1: In conventional gene targeting (A), you must...be included in the final ssDNA template. Figure 2: Overview of ivTRT to generate an ssDNA repair...challenging for sequences with complex secondary structure. Today, multiple companies offer long single-stranded...directs cleavage right before the stop codon!  Figure 3: How to design a C-terminal insertion with Easi-CRISPR... To avoid re-cutting by Cas9 after editing, make sure that the repair template does not contain the wild...facing towards or away from each other.  Figure 4: How to design a conditional allele with Easi-CRISPR...you have a plasmid with your desired insertion feature (e.g., a fluorescent protein), you can amplify ...

...lives of people and society. Future generations of IMP Motivating future generations is key to the success...internationalization become a reality available to all youth, our future, around the world.   In many countries of the world...improvement) and bright students that will be the future of the country. Brain drain is a serious problem...build, together with the universities, a bright future. IMP provides matched one-on-one international ...support to increase the chances of success, but also nurture a supportive and protective environment where individuals...empowered to productively navigate through both their failures and their successes. Although all mentorship relationships...their immediate frontiers. Moreover, sharing a culture/country of origin with an international mentor ...

...expression of kynurenine aminotransferases, to enhance the conversion of kynurenine into kynurenic acid. That...That’s key because, unlike kynurenine, kynurenic acid can’t cross the blood-brain barrier. By lowering...muscles led to another clue: differences in the kynurenine pathway of tryptophan degradation. It turns out...lowering circulating levels of kynurenine in the body, the brain is protected from stress-induced biochemical...brain’s physiological demands,” they write. “In the future, these may lead to treatments for a variety of ...References: Skeletal muscle PGC-1α1 modulates kynurenine metabolism and mediates resilience to stress-...

...pellet for the remainder of the cultures and later maxiprep only the cultures with intact ITRs. I use a spreadsheet...proper packaging of AAV, but also form secondary structures that are prone to deletion from the transfer ...this issue. One way is to grow multiple bacterial cultures of your AAV transfer plasmid at 30 °C instead ...like to miniprep 5 mL of my AAV transfer plasmid cultures and then screen with SmaI digests. I freeze the...PEI transfections. It saves me time and helps me figure out if I have enough plasmid to complete my experiment... viral particles for efficient gene transfer. I figure it’s better to have a bit extra AAV than to have... skip the PEG precipitation of the HEK293 cell culture supernatant because it’s time consuming to prepare...

...synthetases.     Figure 2: Specific binding of scFVs to cognate aaRS antigens as measured by suspension ...encapsulating the protein of interest (Figure 3).   Figure 3: Schematic representation of the MPyV...domain (RBD) of the viral spike protein.   Figure 1. (A) SARS-CoV-2 RBD DMS Library generation and...resolved fluorescence, and suspension bead assay (Figure 2). After finding no cross-reactivity, antibody...within seconds. A CRISPR-based lineage tracing tool captures rare clones that represent 0.001% of the population...found a way to use the accumulation of indels to measure time. These plasmids with different promoters driving...anti-CRISPRs were identified that inhibit the S. aureus Cas9, a Cas protein that is smaller and easier ...

...enabling microscopes to visualize the cellular architecture of whole tissues in 3D with unprecedented detail...thin sheet to illuminate a fluorescent sample and captured the reflected light using a different objective...Ultramicroscope was developed using a stereomicroscope to capture large field-of-views (6). For more comprehensive... on several other LSFM configurations’ various features see (4,7).   Light sheet fluorescence microscopy...methods will greatly aid our understanding of the structure-function relationship of the central nervous system...Jährling, C.P. Mauch,  K. Deininger, et al. (2007). Nature Methods, 4, 331–336. Pubmed. P. Keller, M.B. Ahrens...Neuron, 85, 462-483. Pubmed. E.H.K. Stelzer. (2015). Nature Methods, 12, 23–26. Pubmed. M. Weber, M. Mickoleit...

...your experiments. Capture MS Strategies This approach uses immunoprecipitation to capture proteins from a...been planning, it is a good idea to pause and make sure that your antibody has been appropriately validated...recognition of the target antigen. You want to make sure that your antibody binds to what you expect it to...application and available resources.     Figure 1: Table summarizing the IWGAV five proposed antibody...information for some antibodies, which makes it hard to ensure that you have unique antibodies. An additional ...and then uses mass spectrometry to identify the captured proteins. If the antibody is specific, then you...approach is ideal for validating antibodies for immunocapture applications and is amenable to high-throughput...

...correspond to publications of popular papers. Figure 1: Plasmid requests by date. Parentheses depict...observed between Jan. 1 and Mar. 1, 2020. Figure 2: A simple approximation of the effect of COVID...rough "COVID-19 effect" on order data, it does not capture the fact that different parts of the world encountered...='31668202421', style='text-align: right;' %} Figure 3. An animated map showing the plasmid orders and...during weekends and significant holidays. Figure 4: Plasmid request trajectories suggest an exponential...data likelihood with multivariate Gauss-Hermite quadrature, since the analytical expression for the data...winter break, and COVID-19 on plasmid requests Figure 5: Inferred effects. Values are the inferred percentage...

... the fluorescence amplitude measured for each droplet in each well (Figure 2). A clean ddPCR should have...rate. A rate of 2C/s is recommended to ensure an even temperature change among all of the droplets, but...what if we could repeat this assay 10,000 times to ensure that our titers were as accurate as possible? This...digital PCR involves partitioning a PCR reaction mixture into approximately 20,000 droplets using water-...containing thousands of droplets) can be sufficient to capture the information needed for your PCR experiment....droplet oil that you use should come from the same manufacturer. Otherwise you will end up with poor droplet...reader extracts the droplets from the plate and measures the fluorescence amplitude of each one. Droplets...

...plate to start liquid cultures. Lastly, you can start small overnight liquid cultures with the clones you...use depends on your preferences. Either way, make sure to test your colony PCR primers before using them...will need to screen.   Saving clones for later culture: At this point, you will want to hang onto your...bacteria-water suspension and use them to start cultures of your positive clones. If you want to store ...ones. Regardless of which method you choose, make sure to use the appropriate antibiotic for selection.... on an agarose gel to determine their size. Make sure to use an appropriate molecular weight standard ...samples before pipetting them onto the gel. The figure above summarizes generalized expected results for...

... Figure 1: Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Figure from Zetsche...is also much simpler in structure than Cas9’s; only a short stem-loop structure in the direct repeat region...RNAs important for Cpf1 function, they found that mature crRNAs for Cpf1-mediated cleavage are 42-44 nucleotides.... With this information, they turned to a cell culture system to see if any Cpf1 nucleases would exhibit... its proximity to the cleavage site, preventing future edits. In contrast, since Cpf1 cleaves relatively...deletions at the 3’ end of the target sequence. Figure 2: Effects of (A) double and (B) single base pair...Mismatch positions are indicated below each graph. Figure from Kleinstiver et al., 2016. Cpf1’s strength...

... way to grow haywire, access and create more vasculature to feed themselves, use the blood stream to commute..., introduced the “Three Es of Immunoediting” nomenclature. 1. Eliminate - Latent tumors are recognized...the host immune mechanisms altogether and escape future immune surveillance. Dormancy of certain cancers... system to the presence of cancer are unable to mature into effector cells in the tumor microenvironment... look at a variety of up and coming therapies. Future directions and open questions in cancer treatment...constantly in flux and is dependent on the selective pressures placed upon it by its environment and the body...immune privilege and predicts reduced survival." Nature medicine 10.9 (2004): 942-949. PubMed PMID: 15322536...

...sites targeted by specific sgRNAs. Its RNA-guided nature makes CRISPRa much simpler to implement in genome-wide...transcriptional activator to each genome-bound dCas9 (see Figure 1a for the transgenic construct). More importantly...AAV or by mating with Cre-expressing mice. Figure 1: (A) Schematic of the Cre-dependent SPH construct...probe gene-function relationships in defined substructures of the CNS as well as individual neuronal populations...neurons) and Synapsin-1 (pan-neuronal) promoters (Figure 1b), and sgRNAs can be found here. Neuron-optimized...) with neuron-specific expression of dCas9-VPR (Figure 2), a popular Cas9 activator developed by the Church...including ubiquitous ones such as EF-1α and CAG. Figure 2: Dual lentivirus system with ubiquitous expression...

...is to ensure the propagation of themselves and their associated genes. These systems better ensure that...that plasmids are passed down through generations (Figure 1). These so called “plasmid addiction systems”...the mazF gene and a gene for Zeocin resistance (Figure 2). This MazF-ZeoR cassette is flanked by two direct... repeats. In cells where recombination has not occured, methanol induces the production of the toxic MazF...the mok SD resulting in a double stranded RNA structure that is recognized and cleaved by RNaseIII resulting...bioluminescence reporter in vivo in the absence of selective pressure and found that plasmids were maintained at 100%...with plasmid addiction systems While selfish in nature, these systems have been adapted by researchers...

...identify a few of the features that confer broad host range in plasmids in nature, and provide tips on ... identified some of the features that confer broad host range. These features are: The plasmid origin...characteristics of natural plasmids and how they function in nature can inform us on how to create better recombinant...on how we can harness those features to create broad host range recombinant vectors.  A plasmid’s host...are versatile and adaptable. Variation in ori structure allows plasmids to utilize diverse host replication...and reduced metabolic load (Meyer 2009). Figure 1: Idealized examples of a narrow host range plasmid...multiple oris that vary in DnaA-box and iteron structure, host-independent   Building broad host ...

... quality figure, produce the figure, its legend, and write a results section for that figure. Writing ...next manuscript 1. Be consistent with your figures The figures of a manuscript are the result of a lot of... to my PI about becoming a better writer, I was unsure if I should possibly take a composition class in... individual sections of a paper, the abstract, figures, introduction, etc. so as to thoroughly explain...quilt with your lab mates, you add the individual figures and results to make the final product with an interesting...Scientific Manuscript Writing Scientific Manuscripts: Literature Searching, Reading, & Organizing Write for the...

... identify. Figure 2: Users can cross-interrogate screens from the literature for a drug, disease...particular screens of interest.   Figure 3: Each publication featured in BioGRID ORCS has a dedicated summary...large-scale datasets by computational biologists. Figure 1: The BioGRID Open Repository of CRISPR Screens...compiling genome-wide datasets, which are often obscured in large supplementary data files, BioGRID ORCS.... Importantly, metadata for each screen is captured by BioGRID curators, including the cell line/tissue...determine the gene hits for each screen are also captured as metadata.   Screen results are reported as ...comprehensive pre-compiled downloads and via a full-featured REST Web Service API. Both options are fully open...