...However, there are instances where traditional base editors are preferred. For instance, if target nucleotides...genome editing technique that mediates targeted insertions, deletions, and all possible base-to-base conversions...pegRNA) that both specifies the target site and contains the desired edit (or edits) engages the prime ...combined with any of the PE2-PE5 strategies. PEmax contains a reverse transcriptase with a sequence optimized...strategies to evolve more effective prime editor proteins, but found that the mutations that emerged depended... PE6a and PE6b are small prime editors with RT domains derived from the E. coli Ec48 retron RT and the... ✓ ✓ ✓ Evolved reverse transcriptase domains Smaller size and/or improved efficiency   ...

...developed class of tools are Luminopsins (LMOs), or luminescent opsins: fusion proteins of a light-emitting luciferase...specific biological processes. Addgene’s collection contains two main classes of chemogenetic receptors: Designer...muscarinic receptors and couple to the Gi, Gq, or Gs G-proteins, respectively. KORD is derived from the κ-opoid...engineered α7 nicotinic acetylcholine receptor (nAChR) domains that respond specifically to small molecules termed...chemogenetics plasmids. ID Plasmid Description Gene/Insert Vector Type Promoter Tags PI Do you have suggestions...

...fluorescent markers and tag your proteins of interest with fluorescent proteins in yeast....create fusions of proteins of interest with yeast codon-optimized fluorescent proteins. These vectors are...collection of yeast codon-optimized fluorescent proteins (FPs) in a variety of plasmids allows for constitutive...promoters and the KanMX marker. ID Plasmid Gene/Insert Selectable Marker Publication Additional Resources...Teusink B. In vivo characterisation of fluorescent proteins in budding yeast. Sci Rep. 2019 Feb 19;9(1):2234...

...Some bacteria possess plasmid-borne toxins like colicins and subtilisins to kill other bacteria in the area...microbial defense Genes allowing bacteria to survive toxins are the clearest example of a plasmid benefiting...Conveniently, plasmids carrying genes for bacterial toxins like colicin typically also carry the necessary... the host immune system, and those that produce toxins that cause disease. While these plasmids can be...DNA and other molecules to host organisms. For instance, vectors derived from the Agrobacterium Ti virulence... resistance to that toxin, CcdA. Knowing which strains of E. coli contain this F plasmid is critical for...about some other common plasmid features, such as origins of replication and promoters, check out our other...

...markers. Need fewer components? A spacer can be inserted instead, allowing everything to stay in the correct...spontaneous insertion)   Drosophilia Plasmid pRDB_053 (for transgenic flies; gypsy insulator sequences... the right CRISPR vectors on Addgene and found instead ones that were… pretty close, or at least close...approach, it’s easy to switch out reporters, Cas proteins, guide cassettes, promoters, N’ and/or C’ terminus...various delivery methods, along with compatible inserts. All destination vectors contain an ampicillin ...also why you can’t skip a module entirely, but instead will have to replace it with a spacer.) Each component...expression) Mammalian Plasmid pRDA_791 (contains only orio and ampR) Drosophilia Plasmid...

...difluoride (PVDF) membrane, which is sturdier and retains the blotted protein better than a nitrocellulose...methods. If you are planning to detect multiple proteins through several rounds of stripping and reprobing...membranes recommend re-wetting with methanol before rinsing with wash buffer, as dry PVDF won’t evenly re-absorb... buffer. Be sure to check the manufacturer’s instructions for the membrane you are using. Dry nitrocellulose...20. Once your solution is made, you’ll want to: Rinse the membrane in water or fresh buffer. Cover the...bad!), so be sure to use it under a fume hood. Rinse the membrane in water or fresh buffer. Cover the...imaging step to check if any secondary antibody remains, whether that involves adding fresh chemiluminescent...

... 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...

...and identified interactions of these proteins with human proteins. Their deposited plasmids expressed ...over 75 countries. Figure 1: SARS-CoV-2 proteins and other components. Image from Maya Kostman ...infection and how to interfere with it in a BSL-2 lab instead of a BSL-3 lab. Jesse Bloom’s lab published a protocol...receptor for SARS-CoV. The lab also found that cathepsins play a role in SARS-CoV infection and deposited...Biering et al., 2021). In their search, they found mucins have an important role in viral entry, which are...viral infection.  Expression of SARS-CoV-2 viral proteins In the early weeks of the COVID-19 pandemic, the...expressed 26 of the 29 SARS-CoV-2 proteins which all but one are cloned into pLVX vectors (for lentiviral packaging...

...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...

...each mutated allele.  Cre-lox expressing strains Cre-lox strains are a common example of mice that are derived... Crossing mice to generate a desired phenotype Instead, genetic engineering should be used to separately...independently into different mice, and then these strains should be crossed to generate double-mutant offspring...experiments. You should also maintain your parental strains throughout this process. In the F1 and later generations...genetically engineered mice with a single mutation or strains that have been crossed, there are many considerations...gene randomly into their genome, some transgenic strains are difficult to breed. Some problems with transgenic...expression level of the transgene. In existing strains of mice, the transgene can be maintained on a hemizygous...

...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...

...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...

.... 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...

...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...