...or repression. The TCS light sensors described in ACS Synthetic Biology (2014) represent the newest versions...Photobiology: Optogenetics for E. coli. Schmidl, et al. ACS Synth Biol. 2014 Nov 21;3(11):820-31. Inncredible...compartment (e.g. the plasma membrane, the extracellular space, or the lysosomes) can take many paths. Understanding...that is quick, versatile, and robust. Dubbed the LACE system for light-activated CRISPR-Cas9 effector,...the CRISPR system. The ease and flexibility of the LACE technology makes this system widely accessible for...thereby creating a single-molecule reporter of intracellular processes. Multimerization via the SunTag system...combination of all 11 att pairs in series with unique spacer sequences (pMemoryArray) gives a DNA sequence capable...

...plasmids shipped as agar stabs. Iverson, et al. ACS Synth Bio. 2015. PubMed PMID: 26479688. RAS Pathway...modifiers like DNA methyltransferases or histone deacetylases. Unfortunately, these inhibitors are often non-selective... industry, the bioethanol industry, and the pharmaceutical industry, depend on the yeast, Saccharomyces..., including fermented beer and wine, fuels, pharmaceutical ingredients, and recombinant proteins. In order...

...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...

...with alternating spacers and repeats, was the original impetus for studying CRISPR. Spacers: Copies of foreign...sequences are called protospacers. Repeats: Bacterial DNA that appears between each spacer in the CRISPR array...sequence matches the guide sequence. PAM: The “protospacer-adjacent motif” present in the non-target strand ...the CRISPR array and include a spacer and a repeat sequence. The spacer sequence is the RNA guide, like...real mouthful of a phrase: Clustered Regularly Interspaced Short Palindromic Repeats. That contrast of simplicity...composed of identical repeat sequences and variable spacer sequences. The array is transcribed and processed... into crRNAs, each including one repeat and one spacer. In bacteria, these crRNAs are bound by Cas proteins...

...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...

...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...

...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...

...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...

...antibodies for other sample types and for non-surface (intracellular) antigens. Unfortunately, antibodies frequently... control. This can involve cell surface labeling and/or intracellular labeling (or both). It is important...selectivity for cell surface labeling may become less selective if used in intracellular labeling because ...the intracellular compartment may contain cross-reacting antigens not present on the cell surface. The...best to determine this by testing them. For intracellular targets, we usually test 3 different fix/perms...helpful to try all three when looking to label an intracellular target.   Figure 1: HCT 116 WT and SYT1...optimizing fixation/permeabilization reagents for intracellular targets. Since knockout experiments can be ...

...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’...

...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...