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...Genome-wide Inhibition Library. DNA and cell amounts After deciding on a cell type, you will need to determine...DNA and how many cells to use for your transformation(s). The amount of competent cells you need total ... depending on the particular type of cells you are using, as well as other factors, such as your lab’s...generalized amplification protocols tend not to work as well.   Defining your library First, you’ll need to collect... generally electroporated into electrocompetent cells to ensure the highest possible transformation efficiency...efficiency. Using Stbl4 or other ultra-high efficiency cells ensures that plasmid diversity in the library is...transformations, such as splitting 400 ng of DNA in 100 μL of cells over four individual electroporations. The specifics...

...degraded by the cellular machinery, leaving a small amount of active NgAgo/ssDNA complex in the cell. As proposed...results of Gao et al 2016 in a letter to Protein & Cell. Update (August 3rd, 2017) THE ORIGINAL NgAgo ARTICLE... guide, it cannot be produced from a plasmid in cells. Additionally, in vitro assembly of NgAgo/ssDNA ...dangerous, non-physiological temperature for mammalian cells and one which diminishes the endonuclease activity...guide. Therefore, researchers must co-transfect cells with 5’-P-ssDNA guides and an NgAgo expression plasmid.... Researchers can conveniently transfect/inject cells with in vitro preassembled Cas9/gRNA complexes or...lipofectamine-based method to deliver NgAgo ingredients into the cell, the same method used by Gao et al, 2016 (1). View...

...large amounts of carbohydrates that can inhibit the cell lysis process. To ensure that lysis is complete,...strains adapted for protein purification use the cell’s resources to produce high levels of protein, not...difference? In the simplest of terms, more volume = more cells = more plasmid. Plasmids can vary in copy number...of plasmid copies supported within each bacterial cell in your culture. Copy number is partially specified...richer media or adding supplements can improve your cell density, leading to an improved plasmid DNA yield... supplements to the media You can achieve higher cell density using a nutrient-rich broth such as Terrific...glucose (in moderation) may also serve to increase cell density and plasmid yield. Start with a single, ...

...them. Why Use multicistronic vectors? Detecting cells that are expressing your gene, especially if you...reporters allow you to easily screen or select for cells that are expressing your gene of interest at high...promoter, multicistronic plasmids ensure that any cells that are positive for your marker should also be...anytime you want to express multiple genes in the same cell. Although it is possible to drive co-expression ...promotes translation of the viral genome in eukaryotic cells.1,2 Since then, a variety of IRES sequences have...discovered - many from viruses, but also some from cellular mRNAs. What they all have in common is the ability... cistron due to factors such as the experimental cell type and the specific genes cloned into the vector...

...Table 1 from the paper. 2. Manipulate your cell of interest: Cell-type specific expression of molecular tools...in their cell bodies. Optical stimulation in a different brain area can then reveal the cells bodies of...with AAV: 1. Getting your viral vector into your cell of interest: AAV tropism and route of administration...transduction, the viral capsid proteins bind to the target cell’s surface proteins and this interaction determines...efficiency of the AAV serotype used for a specific cell type. Find some transduction characteristics of ...by restricting expression to genetically defined cell types. Specific transgene expression can be also...different approaches can increase the specificity of cell targeting even further. For example, injecting AAVs...

...for studying the bacterial cell cycle, asymmetric cell division, and cellular differentiation, but it takes...crescentus Multiplexed gRNA for human pluripotent stem cells CRISPR-based DNA demethylation Optogenetic repressors...complex remains in the cytoplasm. Read the paper in Cellular and Molecular Bioengineering Find the optogenetic...Figure 1: Serial dilutions of CRISPRi in Caulobacter cells for ctrA gene expression using various dCas9. Image...popular Streptococcus pyogenes system does not work well in Caulobacter). To test their CRISPRi system, the...lab repressed ctrA, a gene encoding an essential cell cycle regulator. The lab expressed both dCas9 and...piggyBac transposon system for human pluripotent stem cells Lindy Barrett’s lab developed new fluorescently ...

...already available today to cure sickle cell anemia in stem cells but this can’t be used in people until...and how much we have yet to learn. As Dr. Mello said, “Cells have been in the information age for billions...meeting movie stars, but when I met the astronaut Jim Lovell I was speechless (the club of people who have been...genome engineering, Dr. Jennifer Doudna and Dr. Emmanuelle Charpentier. I was joined by Addgene Scientist...from them during a panel discussion with Dr. Craig Mello and Dr. Paul Stoffels from J&J. The panel was moderated...the mechanisms of RNAi, the field for which Craig Mello and Andrew Fire won the Nobel Prize. What did Dr... audience discussed how J&J is already modifying cell and animal genomes to test hypotheses of disease...

...used. The Fujii lab demonstrated this in mammalian cells by expressing dCas9 with an N-terminal 3xFLAG tag...studies (Fujita et al., 2018). In addition to the "in cell" form of this method, in which an engineered DNA-binding...molecule such as the CRISPR complex is expressed in cells to be analyzed, the Fujii lab also developed another...Protein A, 1xFLAG, 2xAM, and HA have been reported, as well as affinity purification with an anti-Cas9 antibody... disease for which there is currently no cure (Campbell et al., 2018). Find plasmids from the Fujii lab...see our interview with Dr. Fujii. References Campbell, Amy E., et al. "NuRD and CAF-1-mediated silencing...recombinant CRISPR ribonucleoproteins." Genes to Cells 21.4 (2016): 370-377. PubMed PMID: 26848818. Fujita...

...make for beautifully compelling photos and videos, while molecular, micro, and cell biology are often more...documentaries. Can you convey enough information to tell a compelling story in an image or do you need a full exhibit...best to. And that's really natural - if it works so well for you, of course you want to share it with others...others! After all, won't it work just as well for everyone else?  In truth, people are incredibly varied...account aimed at octogenarians is going to be a hard sell!) Digital platforms often have demographic data ...should be fun and engaging for the communicator as well as the audience, so pick a mode of communication...Photography Medium (high for microscopy) Visually compelling. Easy to convey a large number of details without...

...into the cells. But for C. roseus, Agrobacterium infiltration into leaves does not work well, as the leaves...tumefaciens strain GV3101(pMP90), which works well for us. Use a well working vector system This allows you to...roots, as functional chloroplasts and leaf specific cell types are missing in roots. The development of transgenic...transformation” usual means that neither a stable cell line or plant is regenerated from the transformed...interest (Unver and Budak, 2009). This method works well in C. roseus. A common method for overexpressing...transient transformation systems for other species as well. Things to consider in developing a transient leaf...fused to a protein of interest to elucidate the subcellular localization of your protein of interest. Find...

...relied on for transducing cells: galactose and the AAV receptor (AAVR). Cell lines lacking galactose were...Concise review: stem cell antigen‐1: expression, function, and enigma." Stem cells 25.6 (2007): 1339-1347... Wilson labs determined LY6A, also known as stem cell antigen-1 (SCA-1), is the receptor used by PHP.B...commonly used to enrich adult mouse hematopoietic stem cells. Its ligand(s) has not been identified. While LY6A...out of Ly6a in primary C57BL/6J brain endothelial cells decreased transduction rates while overexpression...overexpression of the C57BL/6J version of Ly6a in 293T cells enhanced PHP.B transduction rates. The group also determined...PHP.B, virus still reached the brain endothelial cells of these mice, although at lower levels than in ...

...Xenopus frog cell lines to develop robust methods for frog gene editing. They edit in a cell line, then ...is the smallest photosynthetic cell in existence, but most abundant cell on earth (twice the human biomass...Mormyridae (Jason Gallant) Weakly electric fish are excellent systems to study for convergent evolution because...Faithful in 60 °C soils. Dendrobatidae (Lauren O’Connell)  Poison frogs are great for studying parenting... currently for terrestrial frogs. Cnidarian-Dinoflagellate symbionts (Virginia Weis) Sea anemone Aiptasia...killing frogs all over the world. These are less well known fungi (they are not yeast or mushrooms) but...Hawaiian bobtail squid has an extreme number of nerve cells and an unusually high level of RNA recoding in the...

... the same overall process. First users coat a multiwell plate with a capture reagent. The capture reagent...conjugated antibody binds to antigen coated on the multiwell plate. In an indirect ELISA a primary antibody...the primary antibody. In a sandwich ELISA the multiwell plate is first coated with a capture antibody ...antibody can be used. In a competitive ELISA a multiwell plate is coated with a competing antigen (green...enzyme labeled antibody and then added to the multiwell plate. The more antigen in the sample, the less...method, users immobilize the target antigen on the multiwell dish and incubate it with a reporter conjugated...ELISA, the sandwich ELISA, users first coat a multiwell plate with a capture antibody and then add their...

...replicating too quickly can slow cell growth or even kill the host cell. For stringently controlled plasmids...All plasmids rely on their host cell's replication machinery in order to replicate—but not always to ...be synchronized with the replication of the host cell's chromosomal DNA.  Plasmids are said to be under... stringent control when they depend on the host cell’s initiation proteins to start their own replication... occurs at the appropriate time—for example, the cell must have enough nutrients available to complete...the amount of active DnaA available is one of a cell’s best tools to regulate DNA replication. DnaA negatively...available to bind DnaA. Fun fact! As far as we can tell, DnaA doesn’t need to hydrolyze ATP to do its job...

...with enrichment, single-cell sequencing, and optical pooled screens in human cells. Addgene offers vectors...reverse transcriptase enzymes, inhibition of host cell proteins, and modifications to the pegRNA. The Britt...shows enhanced editing efficiencies in a number of cell types at different genomic loci — in some cases,...stabilizing the 3’ end of the pegRNA, which is not well protected by Cas9. This is similar to the proposed...exciting to see future studies enabled by this tool as well as additional potential improvements to the editor... editing efficiencies at different loci in U2OS cells. Image adapted from Yan et al. 2024 under a CC-BY...western blots (Figure 3).      Figure 3: HeLa cells transduced with SARS-CoV-2 nucleocapsid protein ...

... into a pEcCas/pEcgRNA system. This system works well for engineering single genome edits but the double...construct and then introduce both plasmids into the cell. The edit in the chromosome can now take place. .... After editing, pTargetF can be cured from the cell by inducing expression of the sgRNA that targets ...all the edits you want, cure pCas9 by growing the cells at 37 °C.     The new pEcCas/pEcgRNA system We ... to take place. Then plasmids are cured from the cell. Steps 1-1 and 1-2 can be done simultaneously. Image... E. coli B strains, 1 E. coli W strain, and 1 Tatumella citrea, with a total of 70 sites edited. We optimized...crRNA. The spacers of the crRNA can be customized as well, including concatenations into crRNA arrays targeting...

...dynamic samples Ex: monolayers of live cells with fluorescently labelled, motile structures These samples are...high light doses to the cells. Thin static samples Ex: Fixed monolayers of cells or thin (<15 μm) tissue...Biological Imaging and a Lecturer on Molecular and Cellular Biology at Harvard University. No matter whether... 10s of nanometers. Thick dynamic samples Ex: 3D cell cultures, small model embryos >15 μm Thsee are some...shouldn’t be afraid to seek out expertise from a well-established microscopist at your institution when...Biological Imaging and a Lecturer on Molecular and Cellular Biology at Harvard University.     Additional ...similar information References 1. Lichtman JW, Conchello JA. Fluorescence microscopy. Nature methods 2,...

...have become indispensable tools for molecular and cell biologists over the last two decades, but their ...light. Long-term light exposure can also lead to cellular phototoxicity, and experimental success can be...quantum yield, and when transiently expressed in cells, generates much less signal than comparable fluorescent...produce photons. The original nano-lantern emitted yellow-green light and exhibited 3-5 fold increased brightness...three luminescent proteins can be used together in cells. As Nano-lanterns are not subject to photobleaching...phototoxicity, they successfully imaged multiple subcellular compartments over several minutes using Nano-...concentrations of the compound can be added to the media. Cellular esterase then converts diacetyl coelenterazine-h...

...article was written by Alyssa Cecchetelli and Andrew Hempstead. What do smell, taste and sight have in common...with heterotrimeric G proteins on the intracellular side of the cell membrane. Heterotrimeric G proteins...induce or inhibit intracellular signaling cascades as a response to the extracellular stimuli. Ligand dissociation...Peroxidase-Catalyzed Proximity Labeling. Cell 169:338-349.e11 . https://doi.org/10.1016/j.cell.2017.03.028 Roth BL (2016...to the seven alpha helices that transverse the cellular membrane. The receptor recognizes the appropriate...signaling is initiated when a ligand binds to the extracellular surface of the GPCR. This results in a conformational...small molecules to elicit a response in a certain cell type. Chemogenetics is similar to optogenetics but...

...published, followed soon by GCE done in mammalian cells (Shandell et al., 2021).   How it works If you’re interested...with) the cells’ own natural translation machinery; these are often incorporated into the cells via plasmid...so complicated? Well, while the techniques are straightforward, the systems – the cells growing and producing...is usually added to the media for uptake by the cells. Second, you’ll need tRNAs and tRNA synthetases ...biosynthetic pathway for the ncAA integrated into the cell’s genome, instead of incorporation via plasmid or...Theoretically, if you put all these together, the cell will produce a protein with a site specifically ...component of the experiment system (including media, cell type, and growth temperature) may need to be individually...