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...lab and Mustafa Khammash's lab at ETH Zürich. “Instead of a graphical abstract, it’s a musical abstract...turns expression on and off with blue light.   Inspired by @UriAlonWeizmann, here my song about our paper...applications.   Figure 1: With blue light, the VVD domains of the BLADE dimerize and activates transcription...This construct, built from the pBAD33 backbone, contains BLADE, the PBAD promoter, and a mCherry reporter...your gene of interest. pBLADEONLY: This plasmid contains only BLADE and is based on the pTRC99a plasmid...transformed with pBLADE expressing superfolder GFP instead of mCherry under the PBAD promoter. They created...Engineering AraC to make it responsive to light instead of arabinose. Nat Chem Biol. https://doi.org/10.1038...

...which contains the structural and packaging genes, the adenoviral helper plasmid which contains the proteins... to replicate,  and the transfer plasmid which contains the viral genome. In today’s blog post, we’ll...palindromic nature, and high GC content. This instability can cause partial loss of an ITR during routine...constructs in recombination-deficient bacterial strains, such as Stbl3s. ITR loss can be detected by restriction...include a reporter like GFP, some smaller CRISPR-Cas proteins such as SaCas9, CRISPR gRNAs, or your gene of ...

...from Erik Snapp, Associate Professor at Albert Einstein College of Medicine, to learn how problematic ...Research Campus reminds us that, although fluorescent proteins are incredibly useful, chemical fluorophores can...Zika virus and, upon binding, produce reporter proteins. These toehold switches can be used to quickly...and Alexander Green (both formerly at the Wyss Institute) to learn how these amazing tools may be used ...different pH’s and some that are fused to fluorescent proteins. She also showed that R-bodies can rupture E. ... but, even with CRISPR, it isn’t always easy to insert a section of DNA into the genome of your target...Casilio, a system that uses modular RNA binding proteins, dCas9, and a modified gRNA to direct a variety...

...comprised of two tandem fluorescent proteins: a relatively pH-insensitive RFP variant, DsRed.T3, that’s connected...biosensor contains mRFP and GFP fused to the autophagy-related microtubule-associated proteins 1A/1B light...lysosome, and degradation of autophagic substrates inside the lysosome. Historically, methods for studying...the absence of autophagy, the untargeted Rosella remains in the neutral pH cytoplasm where it emits red ...PE due to a C-terminal glycine deletion, so it remains in the cytosol acting as an internal control. Autophagic...

... well characterised antibodies against all human extracellular proteins with community-based validation...18:45 - Reagent Development - Steven C. Almo, Institute for Protein Innovation  1:18:45 - 2:01:18 (end...have a reproducibility “crisis” on our hands and instead provide a brief conceptual framework for the causes...ll see in Steve Almo’s talk about work at the Institute for Protein Innovation (IPI) (49:45 - 1:18:45)...while FPbase curates information about fluorescent proteins so you can best apply these technologies to your...formal systems, individual labs can certainly institute better experimental design practices including...Scientific Figures into Dashboards and Share Your Insights Who gives a tweet? 9 facts about scientists on...

...Last updated on Oct 1, 2020 by Aliyah Weinstein. This post was contributed by guest bloggers, Wenning...into the mouse zygote (Qin et al., 2015).  When inside the zygotes, the gRNA will seek out its target ...pathway (HDR), be it a single nucleotide change, insertion of a reporter gene, or replacement of the murine...case, we saw that 2 out of 3 mice carried a 5 kb insertion in the ROSA locus as assessed by Southern blot...conventional methods. First, one can work with almost any strains of mice, as compared with conventional gene targeting...targeting, which is limited to a few strains, including 129 and C57BL/6, for which we have germline competent...with its human ortholog (greater than 5 kb), it remains to be seen whether CRISPR is as robust as conventional...

...constructs with AAV. In the case of channelrhodopsins, fewer channel proteins are required for the transduced...these tubes. Rinse off the filter - When using a concentration filter column, make sure to rinse off the surface...our Concentrating AAV Protocol (Step 10) for instructions on how to do this. Concentrating your virus ...necessary to visualize expression of fluorescent proteins, for example, as many viral particles must infect...cell to become light responsive than fluorescent proteins required to get a good fluorescent signal. It ...

...the Broad Institute here Read other blog posts about CRISPR Learn more about the Broad Institute and the...the Genetic Perturbation Platform at the Broad Institute and has worked with many Addgenies to help improve...research is still relevant to his work at the Broad Institute today, where he helps researchers in a variety...own research questions. Starting at the Broad Institute For his post-doc, Doench worked on using RNAi... School and then applied to work at the Broad Institute: “The model that the Broad was building that had...development under the Platform model at the Broad Institute because they are “not just developing technology...

...variety of cargo, and even handle a variety of terrains. They are, in fact, a choice delivery vector from...permanently. Viral vectors can also be used to express proteins. Viral expression cassettes typically contain ...from scratch! Why deliver these systems virally instead of by another method? Viral delivery is attractive...thousands of elements simultaneously in your system instead of just one at a time. Many of these libraries ... are packaged and delivered with viral vectors instead of a traditional transfection method. This is because...the actual virus to which immunity is desired. Instead, components of the virus are now delivered in other...immunogenicity of attenuated, nef-deleted HIV-1 strains in vivo. Retrovirology. 2007 Sep 23;4:66. doi: ...

...geneticists to study gene function. In this final installment, I’ll provide a brief introduction as to how ...overexpression and knockdown. Addgene also has a number of insect vectors to consider. An easy way to see what is...available is to browse Addgene’s Collection under the insect expression system or by filtering by fly as species...which locus your transgene ends up (1): Random insertion has the advantage that you can get varying expression... inherited on the same chromosome. Targeted Insertion allows for direct comparisons between lines expressing...would you like your flies ready for use? Random insertions take longer because you need to determine in ...chromosome lines but is not necessary with targeted insertions (here you would know beforehand on which chromosome...

...packaging (Gag, Pol, and Env), the isoforms of these proteins, as well as the viral LTRs, differ. As a result...developed by Garry Nolan at Stanford (Swift, 2001) contains Gag-Pol and either an ecotropic envelope, Phoenix-ECO... just a few days. Another variant, Phoenix-gp, contains only Gag-Pol and enables additional flexibility...transduction are not encoded by the transfer plasmid, but instead are provided in trans by other plasmids or the ...Cloning capacity between the LTRs is ∼8.5 kb, but inserts bigger than ∼3 kb are packaged less efficiently...found at both the 5' and 3' ends of the provirus). Contains sequences necessary for activation of viral genomic... Acts as an RNA pol II promoter. The transcript begins, by definition, at the beginning of R, is capped...

...take a look at these different methods of DNA insertion. Transformation Transformation is the uptake ...inject their genetic material into the cell. Once inside, phages can follow one of two different life cycles...particular integration site. The integrated phage remains dormant until it is triggered to enter the lytic...referred to as transduction. Transferred DNA once inside the infected bacterium can either exist as transient...commonly use phagemids, a DNA cloning vector that contains both bacteriophage and plasmid properties. The...recombination between two nutritional deficient E. coli strains that resulted in a wild type E. coli (Griffiths...bacterium through direct contact. The donor bacterium contains a DNA sequence called the Fertility factor (F-...

...students in real-world lab activities? I’m trying to inspire them and also keep up with all the new biotechnology...being asked in MIT’s research labs, BioBuilder inspires students and teachers to apply engineering principles...final project. BioBuilder’s open-access curriculum begins with students reading comic strips and watching...droppers, centrifuge tubes, circuit parts, and lab-strains of bacteria to study — to conduct their hands-on...breakdown, production of fire-resistant housing insulation, and cleaning up salt runoff from winter icing.BioBuilderClub...summer teacher professional development program that trains nearly 100 teachers each year, with workshops held...BioBuilder's open-access curriculum available on Amazon Inspire.    Many thanks to our guest blogger, Mary Tamer...

...Kaustubh Kishor Jadhav, a Research Assistant at MGMs Institute of Biosciences and Technology. If you are reading...subculturing processes or from the air if the cell line remains exposed for a longer time. The aerosols are invisible... be detected by a luminometer. Non-specific DNA stains can be added in the infected culture medium to ... DNA staining alternative. This method uses DNA stains like DAPI and Hoechst 33258 to stain all DNA. Some...sneezing or coughing in the lab. The human mouth contains a wide variety of mycoplasmas and these can enter... sure to change the gloves and masks regularly instead of keeping them in the pockets of your lab coats...you keep all the required equipment and reagents inside the laminar airflow before and while you are working...

...Genetically encoded voltage indicators (GEVIs) are proteins that detect and output the voltage changes in ...green-fluorescent GEVIs and blue-light-sensitive channelrhodopsins. Find the Ace-mScarlet voltage indicator plasmids...of the MoClo system in cyanobacteria. This kit contains 96 parts including terminators and promoters, ...half with PhyB or PIF6, phytochrome-like fusion proteins from the plant Arabidopsis thaliana. In the presence... light or PCB is no longer present, the fusion proteins separate, inactivating Cre. This is critical for...this system, cells express various fluorescent proteins controlled by Cre recombination. Each cell can...colors depending on the combination of fluorescent proteins expressed. CreLite-mediated recombination was ...

...produced in animals instead of via recombinant DNA, they can’t be genetically tagged and instead are conjugated...reporter consists of two chicken IgY domains. Chicken IgY domains are especially useful for multi-color...’t require a secondary antibody for detection. Instead they are detected with tyramide signal amplification...antibody and a ≥100-fold increase in signal over intrinsic fluorescence. Increased signal is likely due to... et al can be found here. Can’t find a RANbody against your favorite protein? No worries! As more nanobodies...

...target pathogenic proteins, as well as in the lab to deplete expression of proteins of interest. RNAi ...found (typically RNA that the RNAi is designed against), RISC can repress translation and/or degrade target...agriculture as species-specific pesticides and insecticides, an advancement over previous chemical versions... these structures can in turn lead to genome instability. Thus, it’s important to strike a balance between..., I. X., Grunseich, C., Fox, J., et al., Human proteins that interact with RNA/DNA hybrids. Genome Res...10.1101/gr.237362.118 Chen, G. Y., Hur, S. Cellular origins of dsRNA, their recognition and consequences. Nat...

...funding from a variety of government and academic institutions. New graduate students may assume that their...given problem, Empowers the writer, And offers insight into a career as a Professor. Step 1: Your ... to read and fully understand the RFA. The RFA contains all of the important information required for ...the mission of the application and the funding institution. Moreover, avoidance of ‘to be’ verbs creates...time to review the literature and offer educated insight into what the desirable features of future technologies... with the description of the RFA and funding institution. Emphasizing how your solution fits into the ...funded proposal typically lists the deliverables, explains 1-2 potential pitfalls of the intended protocol...

...abilities, we’re gaining new ground in the fight against cancer in its many forms. The dichotomous interactions...major histocompatibility complex (MHC) class I proteins on their surfaces to avoid recognition by CD8+...formation and metastasis, block the cell-surface proteins that help escape immuno-surveillance, and curb...Antibodies that block cell-surface tolerance-producing proteins are one of the most commonly used immunotherapeutic...sequence of immune reactions that aid in the fight against cancer. Sipuleucel-T by the Dendreon Corporation...genome engineering have played a huge role. For instance, researchers are genetically altering tumor cells...cells and immune cells to knockout proteins involved in immune tolerance. Recently, China spearheaded ...

...ensuring that the plasmid they ordered contains the correct inserts, tags, promoters, and other critical...markers, and fusion proteins listed for each plasmid  If the plasmids pass all inspection points, they are...ensure our assembly result is accurate The gene/insert or any major feature highlighted by the deposited...called Geneious. Here, the raw reads are mapped against a reference sequence, if one was provided. If no...assembly in Geneious can still provide additional insights to resolve the assembly and determine if there...