...1,000 active makerspaces around the world. Hackerspaces.org maintains a list of active spaces near you. ... can find a makerspace somewhere near you. The place to start looking for your local space is the Hackerspace...@derekja  Resources Hackerspace Website https://wiki.hackerspaces.org/Hackerspaces OpenWorm Project Website...manifested itself in the creation of Hackerspaces and Makerspaces back in 2007, which function as centres...with the OpenWorm Project One such space is the Victoria Makerspace in Canada, which I belong to. The ...  Funding Makerspaces One of the main challenges when starting a new project at a makerspace is finding...competition. In 2010, he founded makerspace.ca, Victoria’s first hackerspace which now offers members and ...

... take a look at the RSpace ELN.  The connected approach in action: how the RSpace ELN supports reproducibility...import them into RSpace. The kind of informal conversations about research that can take place in Slack normally...repositories. RSpace has been integrated with two of the five: Figshare and Dataverse, as well as the DSpace repository... reproduce an experiment documented in RSpace using the RSpace datasets deposited in Figshare or Dataverse...Rory is founder and CEO of Research Space, which provides the RSpace electronic lab notebook. Rory is based...contributed by Rory Macneil, founder of Research Space. There are many types of electronic lab notebooks...reproducibility RSpace was designed in response to requirements that emerged from intensive engagement with...

...which makes them capable of diffusing in the extracellular space; they also have low immunogenicity and cellular...anterograde tracer and injected at several hundred sites across the entire mouse brain. Traced neurons expressed...it’s less likely to be sequestered in the extracellular space. In addition, rAAV2-retro’s altered amino...particles will diffuse long distances within the extracellular space along an axon, this does not seem to be the...brain tissue is injected with compounds that intracellularly spread away from the site of application and...suggest that there are several parallel mechanisms in place (for review see Maday et al., 2014). It is currently...Goshgarian, 2015). Nevertheless, the use of these tracers over the past few decades was essential to build...

...provided that target is upstream (5') of a protospacer adjacent motif (PAM) Cas9 endonuclease binding to... and a 3-base pair sequence known as the Protospacer Adjacent Motif or PAM. In order for dsDNA to be cut...that each direct repeat, combined with its adjacent spacer, ultimately encodes a single crRNA. The direct...NNGRR(N)  Acidaminococcus sp. (AsCpf1) and Lachnospiraceae bacterium (LbCpf1) 5' TTTV AsCpf1 RR variant... and the unique stretches of DNA between them “spacers” (see figure below). After years of research, we...pre-crRNA into mature crRNA and tracrRNA binding. The spacer regions, on the other hand, are the unique, foreign...tracrRNA forms the scaffold portion of a gRNA and the “spacer region” forms the target sequence.   Overview...

...question I have is, how did we get here in the first place? How CRISPR innovation affected software development...think we are currently at in the CRISPR software space. The CRISPR research community hasn't had a chance...review process has been challenged with many closely spaced CRISPR software submissions. With few prior publications... most value? This is only one of many decisions faced by those engaging in CRISPR design, but it is a ...significant hurdle made worse by an ever increasing marketplace. Since 2013 about 11 tools have been added every...given the lack of attention to it, it is an obvious place to focus further development toward. Quality control...main goals of the software and should be the first place you look to determine a tool’s suitability. Functions...

...Gairik Sachdeva. Healthcare consulting is a fast-paced field, requiring people who are willing to quickly... consulting? Healthcare consultants advise pharmaceutical companies, biotech start-ups, health insurance... commercial challenges or questions that clients face imminently. Consulting firms deploy case teams, ...all parts of a company to collaborate whenever it faces an important challenge. The types of projects mentioned...uncertainties and a rapidly evolving healthcare space. The consultants who enjoy their jobs most, get ...high levels of performance, and occasionally being faced with “shifting goal posts” as a part of a case, ...consulting can sometimes be challenging. The fast paced nature of projects often means working long hours...

...difference. Traces The Traces tab shows the edited and control, non-edited Sanger traces in the region...excluding the PAM sequence. PAM Sequence - The Protospacer Adjacent Motif (PAM) sequence for the nuclease used...as determined by comparing the edited trace to the control trace. In the ICE algorithm, potential editing...and Sanger sequenced. ICE compares these sequence traces to give a detailed analysis of CRISPR editing. ...three tabs - Contributions, Indel Distribution and Traces - provides particular details about the indel profile...the reference sequence derived from the control trace file. On the plot, the green (edited sample) and...calls from the .ab1 file are also shown above each trace. The horizontal black underlined region represents...

...staining both cell surface and intracellular proteins in parallel, then stain the surface protein first, ... Figure 1: Cells expressing a particular surface receptor are stained with a fluorescent antibody...antibodies. It’s also particularly useful for intracellular staining where binding of large immune complexes...antibody thus amplifying the signal.       Intracellular protein targets, measured by fixing and permeabilizing...permeabilizing membranes For intracellular protein targets, users must first fix proteins within the cells...with formaldehyde first to freeze everything in place followed by alcohol permeabilization. Users should..., fix and permeabilize, and then stain the intracellular target. When using this approach, avoid fixation-sensitive...

...photo of an iodixanol gradient after ultracentrifugation. The interfaces between layers are muddled due to...the use of alt text to describe images in online spaces. Alt text is used by screen readers to provide ...from AAV Purification by Iodixanol Gradient Ultracentrifugation First, you’ll want to identify the reader...for AAV Purification by Iodixanol Gradient Ultracentrifugation. The reader may be, for example, a graduate...between a gradient tube before and after ultracentrifugation so that a scientist can extract virus from...details of collecting the purified AAV–that the interfaces between layers are blurred after centrifugation...the syringe needle is inserted bevel-up at the interface between the 40% and 60% layers. Putting them all...

... 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’...

...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...

...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...presented in Figure 2. 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...

...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 ...

...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...

...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...