...Epigenetic engineering with single-pot assembly By Eric Perkins Though it’s easy enough to contemplate DNA in terms... histone-modifying enzymes and reader-effector proteins. b) Golden Gate assembly (backbones available ... create multiple synthetic chromatin-modifying proteins quickly and accurately.   Priode and Haynes, ... is a new tool developed by Rui Tang in Monte Winslow’s lab to label cells after cell-cell interactions...C. elegans since 2015 to conditionally degrade proteins of interest in cells (Zhang et al., 2015). Recently.... TIR1 interacts with endogenous Skp1 and Cul3 proteins to form a SCF E3 ubiquitin ligase complex. In ...protein degradation the Ward lab used fluorescent proteins (FPs) fused to the AID* (minimal 44 amino acids...

... lab characterized two Cpf1 nucleases, distant cousins of well-known Cas9. Cpf1 cleaves DNA in a staggered...indicated below each graph. Figure adapted from Kleinstiver et al., 2016. Until recently, two major elements...off-target editing efficiency. Kim et al. and Kleinstiver et al. characterized genome-wide editing efficiency... to single mismatches, but variably so, with Kleinstiver et al. reporting that Cpf1 can tolerate mismatches...essential function in Cpf1-mediated editing, as Kleinstiver et al. saw no decrease in Cpf1 activity with ...activity than that previously observed with SpCas9. Kleinstiver et al. suggest that AsCpf1’s off-target rate ...eSpCas9 and SpCas9-HF1. Both AsCpf1 and LbCpf1 ribonucleoproteins (RNPs) failed to induce off-target editing...

...AnltWfKuQg pic.twitter.com/OOrwo9feyJ — BTC Institute (@BTCInstitute1) December 23, 2020   Deck the Lab: ...no other, with many scientists working remotely instead of from the lab. Nonetheless, Addgene’s annual ...showcase it all!           View this post on Instagram                       A post shared...TarlingVallim) December 19, 2020 In third place, the BTC Institute decorated with dozens of individual virus snowflakes... to #DeckTheLab with virus snowflakes from @CVRHutchinson #SciArt #STEAM #WinterWonderlab @Addgene Make...Congratulations to the winners! We’ll DM you with instructions to claim your prize. And thank you to everyone...  Aliyah Weinstein...

... at the National Institutes of Health, was an Associate Professor at Albert Einstein College of Medicine...Hughes Medical Institute. His interests include the quality control of secretory proteins in the Endoplasmic... Research Campus of the Howard Hughes Medical Institute. Today's postdoctoral fellows (PFs) face a number...fellowship lasts five years. Most universities and institutes annually advertise for Group Leader positions...new pieces of data, your talk will probably not inspire you or the audience. Ask yourself: What is the...because the search committee asked someone at my institute about me, even though I had not asked this person...successes can be infrequent and a hobby that provides instant gratification can help boost your ego in between...

...difficult to distinguish between proteins bound by the antibody vs proteins bound to the target protein, ...to confirm that your antibody is not binding to proteins outside of your target protein. Show suitability...antibody can distinguish between two very similar proteins. Additionally, they require variable expression...These strategies use multiple, unique antibodies against the same protein, each targeting a different epitope...correlation between the results. Using antibodies against different epitopes on the same target reduces the...This approach uses immunoprecipitation to capture proteins from a sample using the antibody and then uses...uses mass spectrometry to identify the captured proteins. If the antibody is specific, then you expect that...

...useful not only for future studies of kinesins but for many other proteins of interest too! Find CLIP, CLAP...brighter and more photostable than fluorescent proteins, making them ideal for single-molecule fluorescence...FRET to study the intramolecular dynamics of the proteins in solution and when associated with microtubules...microtubules. Figure 2: CLIP-SNAP fusion proteins for single-molecule FRET. A) Schematic of a CLIP-SNAP...flexible linker where a protein of interest can be inserted. B) Protein expression and labeling procedure....deposited an array of constructs for tagging the kinesins KIF5A and KIF5B as well as backbones for generating...generating other SNAPf-, CLIPf-, and CLAP-tagged proteins for mammalian cell expression. These plasmids will...

.... Multiple inserts: There are some cloning methods that make working with multiple inserts a breeze. Among...large number of inserts to assemble. You could also consider using Gateway if your insert number is a bit...another important consideration is: The number of inserts ...as some methods are more amenable to combining...Here you simply PCR-amplify your vector and your insert, DpnI digest away the templates, and transform ...toposisomerase I, and a taq polymerase-generated insert. When mixed, topoisomerase I provides both the ...enzyme mixes. After generating the appropriate inserts by PCR, there is no need for restriction digests... very high chance of success (>95% for a single insert), mostly due to the use of toxic ccdB to prevent...

...experience with my blog helped immensely when CINSO started brainstorming the resources we wanted to provide online...and other nonprofits to gain support for CINSO. For instance, I tweeted to the Indiana State Museum and...nonprofit called Central Indiana Science Outreach, or CINSO. We organize fun science events for adults and professional...called “nontraditional,” helped prepare me to start CINSO. Here, I share some of the tips I learned along ... best supporter and most productive partnership CINSO has had. Volunteer! The turning point in my graduate... and graduate school resources daily to promote CINSO events and recruit speakers and participants for...something I had zero experience with before founding CINSO. Now, the network I built during graduate school...

...the proteins they label. In fact, a recent study of 400 proteins found that 20% of tagged proteins did... protein must be tested carefully to ensure it retains the attributes of the native protein. Since this...notably high solubility, limited affinity for other proteins, and accessibility to an antibody. From their ...candidates, they selected 12 candidate protein domains from non-vertebrate systems. Preliminary testing...sophisticated interactions with multiple other proteins. Again, little to no change in cell morphology...Series. Get the Inntag Plasmids Tag endogenous proteins with CRISPR Fluorescently Tag Your Protein of ...

... protect you and your institution, and almost all universities and institutions require them for material...protective of both the providing institution and the receiving institution. Therefore, many technology transfer...transfers. The UBMTA was drafted by the National Institutes of Health (NIH) and adopted by the Association...benefits for both the providing and receiving institutions and their respective researchers. Check out ...knowing your rights – and the rights of your institution – are protected while we handle all the paperwork... to you, or we can send it directly to your institution’s MTA coordinator. Organizations that have opted...

...DNA-binding molecule, such as Zinc-finger or TAL proteins, can be used. The Fujii lab demonstrated this ...spectroscopy studies allowed for the identification of proteins associated with the IRF-1 locus. Figure...was an intensive process. One method required insertion of a recognition sequence into the genomic DNA...that do not use dCas9, require Zinc-finger or TAL proteins targeting a specific genomic locus, which can ... addition to the identification of DNA binding proteins, this method can characterize nucleic acid-nucleic... D4Z4 array is modulated by DUX4-induced MBD3L proteins." Elife 7 (2018): e31023. PubMed PMID: 29533181...genomic regions and identification of associated proteins by engineered DNA-binding molecule-mediated chromatin...

...constructing fusion proteins, check out our article: Fluorescent Proteins 101: GFP Fusion Proteins - Making the...factors. For example, FRET requires that fusion proteins be in close proximity and present in large enough...protein-protein interactions that uses split fluorescent proteins tethered to potential protein interaction partners...difficult with FRET, as the overexpression of fusion proteins it requires can lead to aberrant subcellular localization...localization of BiFC constructs should also be compared against those of their endogenous counterparts (by immunocytochemistry...call for a simple, yes-no answer — i.e., do two proteins interact or not? — FRET is preferable when the...Visualization of Interactions among bZIP and Rel Family Proteins in Living Cells Using Bimolecular Fluorescence...

...and understanding of CRISPR, and perhaps provide insights and ideas for your own research! Classification...bacteria and nearly 100% in archaea, though Class 1 remains largely underutilized compared to Class 2. Class...effector complex but are defined by the accessory Cas proteins in the Cascade complex. Despite a tenuous grasp... genes, Cas1 and Cas2, which are critical for inserting short fragments of foreign DNA into the CRISPR...CRISPR in more depth, the Jennifer Doudna lab maintains an encyclopedia of Class 2 CRISPR systems called...a CRISPR-associated transposase (CAST) that can insert large fragments of DNA. The V-F variants, which...discovery algorithms find new candidates for CRISPR proteins. A type VII is already on the horizon (Altae-Tran...

...recombination is the process by which nearly all domains of life repair genomic damage, specifically double...sequence homologous to the chromosomal site of insertion. The linear PCR product is introduced into the...the cell by direct transformation. A given insert will typically contain both a protein modification sequence...imaging studies rely on direct fusion of fluorescent proteins (FPs) to a yeast gene of interest. These fluorescently...abundance, movement, and localization of individual proteins, but also investigate protein-protein interactions...interest, allowing for easy capture and detection of proteins and complexes, without the artifacts sometimes... PCR product from this collection will yield an insert with the format (protease site)-6xGly linker-(epitope...

...cleave affinity tags from purified recombinant proteins at small and large scale.  This new technique ...technique involves selection in the cytosol (instead of on the yeast extracellular surface) which enables tracking...demonstrated to work for evolution of other target proteins and might be useful for your studies.  Find the... Angela Abitua Genetically encoded fluorescent proteins are often used to label  specific structures, ...within cells. However, sometimes these fluorescent proteins might not localize to the right places, which ...to improve nuclear localization of fluorescent proteins, they found that adding a triple nuclear localization...in the cytosol of E. coli, however the nanobody proteins tend to get stuck in inclusion bodies. Takayuki...

...co-repressor important in development. Groucho family proteins are found in flies, worms, frogs, mice, and even...the Shivdasani Lab from the Dana Farber Cancer Institute! Sonic Hedgehog, an important regulator of embryonic...named by a postdoc in Cliff Tabin’s lab who took inspiration from a comic book his daughter was reading. SHH...synthetic transposon system that can be used to insert specific pieces of DNA into vertebrate chromosomes... et al., 1991, Jordan and Karess, 1997), which contains the regulatory regions that are necessary for ...spaghetti? I actually couldn’t find the specific origins of the name but some of our Addgenies had an idea...plasmid in our repository, plasmid 39624, that contains cmr.   tinman and bagpipe, two genes important...

...you produce viral vectors using viral envelope proteins from another virus to either restrict or broaden... but you use a different envelope glycoprotein instead of the wild-type glycoprotein. The pseudotyped ...protein is encoded on the envelope plasmid which contains a promoter, the envelope gene, and a polyA tail...virus:  Lowering cytotoxicity. Different envelope proteins have different levels of cytotoxicity and the ...pseudotyping methods with other viruses in a BSL-2 lab instead of a BSL-3 or BSL-4 lab. For more details, check...in serum (DePolo et al., 2000). Other envelope proteins for pseudotyping While VSV-G is the most commonly...for pseudotyping, there are many more envelope proteins that can be used for specific purposes as described...

...split protein like Cas9 is to use split inteins. Split inteins are a pair of naturally occuring polypeptides...toxicity. More AAV-based CRISPR systems Smaller Cas proteins SaCas9 isn't the only CRISPR enzyme that’s small...advantage that it can be turned off by anti-CRISPR proteins. The Sontheimer lab used NmeCas9 with AAV to edit...disease-causing loci in mouse liver.  Two other smaller Cas proteins include Cas12b and CasX. While there are no papers...papers yet published that deliver these Cas proteins with AAV, at just 1,108 and 986 amino acids, respectively...polypeptides that, when at the ends of two proteins, mediates protein trans-splicing, similar to an intron...developed a split intein spCas9-AAV toolbox that retains the gene-targeting capabilities of full-length ...

...upstream of either NGA or NGC PAM sequences (Kleinstiver et al. 2015). From these screens, they identified...mutations found in the REC2, REC3, and PAM interacting domains and allows for expanded PAM recognition as well...off-target activity (Hu et al., 2018).  In 2020, the Kleinstiver lab reported the development of near-PAMless ...the CRISPR toolbox The isolation of novel CRISPR proteins has and will continue to dramatically increase...which makes it easier to target AT-rich genomes (Kleinstiver et al., 2016). Type VI CRISPR systems, which ...Wolfe SA, Sontheimer EJ (2018) NmeCas9 is an intrinsically high-fidelity genome-editing platform. Genome... plasmids from this publication at Addgene. Kleinstiver BP, Prew MS, Tsai SQ, Topkar VV, Nguyen NT, Zheng...

...limited to traditional restriction enzyme cloning. Instead, you can choose a molecular cloning technique that...takes place between the attB sites flanking the insert and the attP sites of the donor vector. This reaction... inhibits the growth of CcdB sensitive E. coli strains, most colonies should contain the desired, recombined...vector.   Method B: TOPO-cloning of the desired insert into an attL-entry-TOPO vector. TOPO cloning adds...Method B to create an entry clone: TOPO cloning the insert into an attL-containing entry vector.   Method...interest and a attL-entry vector. This fragment is inserted in a multiple cloning site (MCS) of an attL-containing... create an entry clone: Restriction cloning the inset into an attL-containing entry vector.   Pro...