...stage acquire genetic alterations and alter cell-surface antigen production to evade the immune system. ...APCs. Furthermore, tumor cells alter their cell-surface proteome to inhibit expression of co-stimulatory...histocompatibility complex (MHC) class I proteins on their surfaces to avoid recognition by CD8+ T cells. Existing...tumor formation and metastasis, block the cell-surface proteins that help escape immuno-surveillance, ...tumor microenvironment. Antibodies that block cell-surface tolerance-producing proteins are one of the most...penetration, inadequate concentration, mutation of cell surface targets by tumor cells, are a few of the reasons...producing chimeric antigen receptors (CARs) on their surfaces have been the most acclaimed form of immunotherapy...

... the location of the ‘A’ relative to the protospacer adjacent motif (PAM). ABE7.10 can edit target 'A'... deaminate cytosine placed in 2-6 bp and 4-8 bp, respectively, of the protospacer. And if the target cytosine...A' if it lies in a small genomic window: at protospacer position 4-8 counting the ‘NGG’ PAM as 21-23 (...closer to the PAM, i.e., positioned at 8-10 in protospacer, ABE6.3 or ABE7.9 are better suited (Gaudelli...two Bsa1 sites to clone the single guide RNA (protospacer+scaffold) in a pol-III (OsU3) promoter driven...tRNA-gRNA (PTG) gene system to clone multiple protospacers which enables us to target more than one genomic...depending on the location of your target base in the protospacer sequence. If you do not find a suitable ‘NGG’...

...The target is present immediately adjacent to a P rotospacer A djacent M otif (PAM) The PAM sequence (NGG...codons that make up a gene PAM P rotospacer A djacent M otif; Sequence adjacent to the target sequence that...referred to as CRISPR ( C lustered R egularly I nterspaced S hort P alindromic R epeat) technologies. Before...for Cas-binding and a user-defined ∼20-nucleotide spacer that defines the genomic target to be modified....through interactions between the gRNA scaffold and surface-exposed positively-charged grooves on Cas9. gRNA... an active, DNA-binding configuration, with the spacer region of the gRNA left free to interact with the...Cas9 will only cleave a given locus if the gRNA spacer sequence shares sufficient homology with the target...

...Integrins are essential cell surface receptors involved in complex signaling pathways and the linkage...linkage of intra- and extracellular environments. Found on nearly every cell in the human body, integrins are...eight beta subunits. Both subunits have short intracellular ends and large ectodomains containing ligand-binding...the cell membrane, physically connecting the extracellular environment and the cytoskeleton. Most integrins...affinity for ligands. When integrins interact with extracellular ligands, their ectodomains can extend and open...mediating cell adhesion and migration, initiating intracellular signal cascades, and allowing agile responses...it is resisted by the ligand embedded in the extracellular environment. This further stabilizes the extended...

...drive sequence replacement in cells. Targeting three dCas9 molecules to a precisely spaced region allowed...approach to improve the efficiency of sequence replacement by HDR. Understanding Cas9 biophysics enhances...studies) to boost the efficiency of sequence replacement in vivo. We discovered that single stranded donor...Richardson et al., 2016.   dCas9 enables sequence replacement without cutting Our in vitro results also demonstrated...allowed sequence replacement rates of approximately 1%. This is by no means a large number, but it was attained...to tackle genetic diseases in which sequence replacement holds some kind of fitness advantage but error-prone...this technique supports gene disruption or gene replacement frequencies that are an order of magnitude greater...

...information from a region (anterograde tracer, Fig. 1B). Tracers are typically delivered to a particular... either trace downstream from the soma (neuron cell body) toward the axon (anterograde tracer) or upstream...neurons that send information to a region (retrograde tracer, Fig. 1A), and another to visualize neurons that...from the axon terminals to the soma (retrograde tracer) (Fig. 2). Viruses as neuronal scalpels Viruses...genetic information or tools, making them useful tracers, but finding a virus that works well for retrograde...that you understand this new tool, go forth and trace some neurons!  This post was originally written ...

...antibiotic-resistant cells; during the time required to grease, place and fill a dozen cloning rings, the remainder of...a 10 cm dish in 15- 20 minutes- a far more rapid pace than using cloning rings. A picture of a 60 mm dish... pipet tips (USA Scientific #1011-8410) is also placed in the hood. Finally, a sterile 2X DMEM solution...bicarbonate) is brought to room temperature and placed in the hood. The medium from a 10 cm dish containing...punched out, gently sucked up into the pipettor, placed into wells, and partially dislodged from the agarose...the plates that do not appear to be yielding well-spaced clones. For rapidly growing cells we sometimes ... TPP peelaway T-75 flasks to provide a greater surface area (the amount of agarose/DMEM should be proportionately...

... a complementary DNA sequence, and (3) a protospacer-adjacent motif (PAM) on the opposite strand. Once... strand is edited by base editors! PAM Protospacer-Adjacent Motif A short sequence that must be present...an —NH2 (amine) group from the target base and replaced it with a double-bonded oxygen. This simple enzymatic...Once it finds a PAM, Cas9 unwinds the adjacent DNA to create a region of separate strands called an R-loop...that preferential C → G editing was possible, the race was on to develop more efficient and precise editors...activation domain (SAD), which recruits histone acetyltransferase to open chromatin, improved editing efficiencies... editing, an alternative editing strategy that replaces a target region with a newly synthesized, templated...

...assembly of biotinylated SpyTag-SpyCatcher linkers and spacer proteins ending with a GFP nanobody (Figure 1)....determination. A) Schematic of workflow. B) Detail of spacer peptides (3HB: 11-nm 3-helix bundle; SAH: 60-nm...software (green circles), magnetic bead (*), and spacers are labeled; scale bar: 100 nm. D) 3D model of ...other targets. The modular system of linkers and spacers could enable many future variations using nanobodies...Cryo-EM samples can be damaged at the air-water interface (AWI) during the freezing process, resulting in...When proteins (blue) interact with the air-water interface (AWI) before vitrification, they can be completely... Small LEA proteins as an effective air-water interface protectant for fragile samples during cryo-EM ...

...mutated allele introduced from a conjugative plasmid replaces the chromosomal copy. This is a versatile technique...modifications to pDS132 (Philippe et al., 2004): Replaced the sacB cassette with one of three toxins from... a broad array of pathogenic Proteobacteria. We placed the toxin genes under control of a rhamnose-inducible...the Salis group) (Espah Borujeni et al., 2014). Replaced the pDS132 MCS with a versatile, synthetic MCS... “pTOX” in a broad group of Gram-negative Enterobacteriaceae including in E. coli O157:H7, Shigella flexneri... point mutations. I think it will always have a place in every bacterial geneticist’s toolbox. Depending...negative selection systems for unmodified Enterobacteriaceae." Nucleic acids research 43.13 (2015): e83...

...new teaching space at Lab Central to learn all about it. LabCentral is a lab and office space shared by ...and making space for the techniques ourselves. As you probably have limited time and space in your own...by numerous emerging biotech companies. The space was modern with clean bold colors of orange and white...like workshops at Lab Central. Because of the fast pace of work in the Addgene lab, we don’t often get the...opportunity to explore new techniques in our own space. Workshops such as this give us a chance to learn...

...many histidine residues to include.  Placement As far as placement goes, there is no cut or dried answer...frequency amino acid, proteins containing two or more adjacent histidines are present in most production systems...polyhistidine tag plasmids here! Where should I place the tag and how many do I need? When designing a...ions once the protein is folded. Sometimes a tag placed on one end will be blocked during protein folding...should try tagging the opposite end.  If neither placement works, consider purifying under denaturing conditions...terminus is preferable over the other however, tag placement can affect protein function. B, The string of ...

... B cells. They bind to proteins on the surface of extracellular pathogens such as parasites or microbes...microbes, or to proteins expressed on the surface of cells that have been infected with a microbe, to trigger...the amount of protein present. The reaction takes place in a 96-well plate, where primary and secondary ...Flow cytometry is used to detect proteins on the surface of or inside whole cells. Antibodies - typically...cells: antibodies can bind to proteins on the cell surface, or the cell membrane can be permeabilized to allow... allow antibodies to bind to intracellular proteins. The fluorescence signal is detected by the flow cytometer... activate cellular pathways by binding to cell surface receptors. These applications are typically used...

...receptors expressed on the cell surface. Thus, a screen can be used to target surface proteins to determine which...known to cause the up-regulation of an important surface protein (e.g. PD-L1 on a cancer cell). In this ...signaling molecules were involved in the phenotype. Surface proteins  My group works on immune cells in different...transferred cells is followed. A benefit of working with surface proteins is also that such proteins are easily ...further be used to interfere with the identified surface proteins, and such antibodies could thus be interesting...all differentially expressed genes that code for surface proteins. One of the first challenges with a custom...the information that you have input in the web interface, making it convenient for you to later identify...

...LanthaScreen) (Degorce et al., 2009), measuring intracellular calcium (Miyawaki, Griesbeck, Heim, & Tsien,... to get your fluorophore delivered to the right place? FRET experiments require targeting the donor and...specificity Donor fluorophores need to go to the right place. Mis-targeted donors never “see” FRET acceptors,... site when used in conjunction with Cy dye-nitrilotriacetic acid (NTA) conjugates (Kapanidis, Ebright,...proteins (such as GPCRs, ion channels, or cell surface receptors) or with experiments on permeabilized...Biarsenical labeling reagents If you want to label intracellular proteins covalently with FRET acceptors, then...and the acceptor fluorophore binding sites are adjacent to each other on the same protein and a negative...

...transsynaptic neuronal tracer into a monosynaptic (first order) neuronal tracer For RABV to crawl through...neurons are connected. Viral Vectors as Neuronal Tracers Due to their ability to efficiently enter cells...However, they generally are not transmitted to adjacent neurons via synapses (i.e., transmitted transsynaptically...cells through their interactions with target cell surface receptors (Callaway 2008). Based on this, cell ... RABV expressing the viral protein EnvA on its surface will only infect Cre-positive, TVA-expressing cells...taken up by the presynaptic nerve terminals of adjacent neurons (i.e., it is taken up by projection neurons...

...better access to intracellular targets. If your target is already on the extracellular side of the membrane...proteins in place, while allowing antibodies into the cell where they could bind to intracellular epitopes... Organic Solvents Organic solvents, generally acetone, methanol, or ethanol, work by dehydrating and ...and leaves many membrane-associated proteins in place. In contrast, Triton X-100 and Tween-20 are examples...is suitable for antibodies that bind to the extracellular portion of a protein. B) Permeabilizing with...

...Lab: Classic Edition Winners In a tie for first place, the Vestibular Lab decorated not just their lab...@druryhannah) The Tarling-Vallim Lab’s first place-winning entry doubled as their lab’s decoration ...Lab (@TarlingVallim) December 19, 2020 In third place, the BTC Institute decorated with dozens of individual...Deck the Lab: Home Edition Winners Winning first place in the home edition, the MicrobiGals created glow...Microbigals (@MicrobiGals) December 23, 2020   In second place, Dr. Anne Madden created a microbe advent tree displaying...Madden (@AnneAMadden) December 23, 2020   In third place is Matthijs Dorst’s Deck the Lab Nativity scene ...

...paragraph is the place to introduce yourself and your background. It’s a great place to state why you’...What is a cover letter for? A cover letter is a place to provide helpful context about your candidacy,...candidacy, beyond what is on your resume. It is a space for you to explain how your skills and experience match...candidate!  Additional uses The cover letter is a great place to proactively address questions about your application...you haven't talked about it yet, it's also a good place to state why you are interested in working for this...applicants. In short, while a cover letter can’t replace skills and experience, it can help the hiring manager...

...Bordetella spp., Caulobacter spp., Enterobacteriaceae, Gluconacetobacter xylinus, Haemophilus influenzae,...Gram-negative and Gram-positive  Acinetobacter calcoaceticus, Actinobacillus pleuropneumoniae, Actinomyces...carotovora, E. chrysanthemi, Escherichia coli, Gluconacetobacter xylinus, Gluconobacter spp., Hyphomicrobium...pSa, pR388 Gram-negative  Acinetobacter calcoaceticus, Aeromonas liquefaciens, A. salmonicida, Agrobacterium...campestris pv. campestris, X. campestris pv. malvacearum, Zymomonas mobilis pBBR1 based pBBR1 ...Caulobacter crescentus, Escherichia coli, Gluconacetobacter xylinus, Paracoccous denitrificans, Pseudomonas...