... both of these cases, transposons combine the advantages of viruses and naked DNA while eliminating some...therapy - avoiding the use of viruses is also important for social and regulatory reasons. 1.) The transposon...Zsuzsanna Izsvak’s lab screened many SB tranposase variants for hyperactivity in mammalian cells. Their efforts...transposase (as determined by transposition assays of antibiotic-resistance genes conducted in HeLa cells). In...inducible expression vectors were developed into 16 variants with different selection markers (blasticidin,...as a nonviral vector for CD19-directed chimeric antigen receptor (CAR) therapy. SB is a valuable tool and...

...findings can be predictably applied to future work. Importantly, no one should expect that every study should...who have complaints about the unreliability of antibodies. IPI is working to address this issue directly...directly by creating well characterised antibodies against all human extracellular proteins with community-...research tools. BenchSci collates information about antibodies while FPbase curates information about fluorescent...and use computer based analysis techniques, an important component of reproducibility is making sure that...figures. Cause #2: Hidden “negative” data We all want to be able to tell a good story when presenting ...journal’s perspective, probably not what most readers want to read. As Susanna Bachle points out in her talk...

...reporter-based) each come with their advantages and disadvantages. Sanger sequencing is simple and the...1B below). Alternatively, if an attB-attP pair is anti-aligned (sites facing opposite directions) on a ...from multiple primer pairs, one can infer the predominant state of registers within a sample using a metric...multiple registers. However, it is only semi-quantitative. It won’t tell you the distribution of states...states in a sample; instead, it will suggest the predominant state of registers in a sample based on a similarity...of inputs (see Figure S9 of our paper (1)). We anticipate that, depending on the context and application...

...expression. Experimental output is measured and quantified. Created with BioRender.com.   Optogenetic...2020). Light-gated ion channels are useful when you want to activate or inhibit specific neurons without ...pathway. Photoswitches are great for teasing out important branch points along a signaling cascade that may... activated. Created with BioRender.com.   Important considerations for using optogenetic tools Once...optogenetics or biosensors in one system. There can be substantial overlap in the activation curves (Marshel et ... fidelity with the stimulus. For example, if you want to stimulate at a fast frequency without missing...can wreak havoc on your experiments, so it is important to understand the details of how each tool performs...

...administered systemically, without causing disease? Recombinant adeno-associated viral vectors, or rAAVs, can...serotypes can be useful to evade neutralizing antibodies developed from prior natural exposure or rAAV...route of administration (Wu, Asokan, et al., 2006). Want to know if a capsid is a good option for your experiment...61779-370-7_6 Tabebordbar, M., Lagerborg, K. A., Stanton, A., King, E. M., Ye, S., Tellez, L., Krunnfusz.... Directed evolution of a family of AAV capsid variants enabling potent muscle-directed gene delivery ...., Gerfen, C. R., Schiller, J., Dudman, J. T., Hantman, A. W., Looger, L. L., Schaffer, D. V., & Karpova...Karpova, A. Y. (2016). A Designer AAV Variant Permits Efficient Retrograde Access to Projection Neurons. ...

...reporter can also quantify Cas9 cleavage, thus exhibiting its versatile utility in quantifying multiple gene...versatile in vivo fluorescent reporter for rapid quantification of base-editing and Cas9 cleavage activity ...several cell types is the lack of a rapid and quantitative editing assay. To help accelerate the expansion...plasmid that allows for rapid fluorescence-based quantification of base editors that utilize dCas9 fused to...frame-shift mutation within the mCherry reporter. Quantification of editing efficiencies simply involves tracking...

...restriction sites are present in a given sequence. You want to choose enzymes that: Do not cut within your...Next, we need to examine the DNA sequence that we want to amplify and design primers that will bind to ...primer design: Because we are cloning an ORF, we want to clone from the start codon (ATG) to the stop ...reaction Run PCR to amplify your insert DNA. It is important to use a high fidelity polymerase to minimize .... The fidelity of the polymerase becomes more important the longer the expected PCR product is. You should...used. No matter which polymerase you use, it is important that you sequence the final product. Additional...

...treated a tetracycline resistant plasmid, pSC101, and a newly developed kanamycin resistant plasmid, pSC102,...with EcoRI and selected for E. coli transformants that were resistant to both. When this proved successful...generic term for any extrachromsomal hereditary determinant”. His proposal, however, was basically ignored...was the tool of choice for molecular biologists wanting to study bacterial genetics. This all changed thanks...

...other professors at the University of Barcelona wanted to develop strategies to make our classes more ...thinking about how to solve this problem. I didn’t want to face another group of sleepy students at 8 a....engaging card game could help me get the engagement I wanted. I therefore started planning a game that would...following lessons. And let me tell you more, they had brilliant ideas on how to solve many therapeutic and pre-clinical...educational nature of this material are welcome and if you want to receive our modifiable powerpoint file, please...University of Barcelona. He is also founder and consultant at FRESCI particularly interested in advanced...

...us take comfort in the fact that it’s often not quantity, but quality that really matters. Well, it turns...another example, neuronal gene expression by an anterograde AAV was shown to drop off drastically with lower...concentrating your AAV above your desired titer and want to dilute it, err on the side of caution when adding...References 1.Clark, K. Reed. "Recent advances in recombinant adeno-associated virus vector production." Kidney...PMID: 11841606. 2. Schultz BR, Chamberlain JS. Recombinant Adeno-associated Virus Transduction and Integration...PMC2574934. 3. Zingg, Brian, et al. "AAV-mediated anterograde transsynaptic tagging: mapping corticocollicular...

...time, he is pursuing a plant engineering project at BosLab. Once he optimizes plant cell culture and transgenesis...transgenesis he wants to share his knowledge with the community by teaching a DIY plant engineering workshop...and to see old pictures of the lab!). By 2016, we wanted to be taken seriously so we made it official by...ecology but she became a member at BosLab because she wants to engineer microbes. We’ve ordered a few yeast ...

...physiologically-relevant systems, to catch all the important interactions. It’s also important to determine...speak, with collaborators in Paris and New York. We want YOU to join the fight against coronavirus We are... a lot of background in our study - so you might want to be careful with these clones (and help us figure...been quite the administrative headache receiving constant email requests and tracking their fulfillment ...

...Imaging by Coat protein Knock-off) instead takes advantage of the ribosome’s movement. When the ribosome ... reporter. Delayed translation is known to be important in development, and Halstead et al. used TRICK...mRNA and keeps it in a single field of view for constant tracking. You can then directly monitor multiple...that can recruit GFP fused to the SunTag-specific antibody scFv. Each time the reporter is translated, the...second. They also explored the effects of 5’ UTR variants in translation of cell cycle protein Emi1, showing...and 3’ UTR. Before beginning experiments, it’s important to verify that these changes do not impact translation...

...This post was contributed by guest bloggers Samantha Zyontz and Neil Thompson from the MIT Sloan School...pulled the journals from 2005 to 2015 in areas most relevant to plasmids: Genetics & Heredity, Cell Biology...after controlling for journal. And so, while our quantitative analysis cannot rule out all other explanations...along the way. Many thanks to our guest bloggers, Samantha Zyontz and Neil Thompson. Special thanks to Addgenies...for helping compile the data used in this work. Samantha Zyontz is a Ph.D. candidate at the MIT Sloan School...ssrn.com/author=1232466.     Neil Thompson is an Assistant Professor of Innovation and Strategy at the MIT...

...people use is the built-in whiteboard, in which participants can contribute and sketch on the board without...control panel on the top of the screen and every participant get to annotate things as they come up in their...time breaks up the monologue a little and gives participants something else to focus on. Use collaborative... creativity are pivotal in writing publications/grants too. As described by several works (Wuchty et al...fades away the longer we are on it – so it is important to take breaks. After 1 hour (or another designated...be done randomly or manually assigning which participants to group together. Breakout Rooms can be used...

...-AAV-syn-jGCaMP7c variant 1513-WPRE (105321-AAV1) pGP-AAV-syn-FLEX-jGCaMP7c variant 1513-WPRE (105322-...vectors to more serotypes. We currently package four variants of these biosensors, which are optimized for specific...relative to baseline fluorescence, as the GCaMP7f variant, and is approximately 5-fold brighter than GCaMP6s...GCaMP6s. jGCaMP7f is the fastest responding variant. jGCaMP7b exhibits the brightest resting fluorescence...fluorescence from inactive neurons is reduced. This variant has an approximately 12-fold increase in brightness...

... mAID and expressing the essential plant protein TIR1 in non-plant cells, which interacts with the cells...Cecchetelli Auxin Inducible Degradation (AID) uses plant hormones called auxins to conditionally and reversibly...a degron, a domain that induces degradation. In plants, auxin binds to TIR1 of the SCF-TIR1 ubiquitin ...engineering SpCas9 Article contributed by Shreya Vedantam CRISPR/Cas9 is widely used genome editing tool...versatility of the engineered SpCas9-NG and its variants can be used to engineer specific sites that were...

...expression and largely defines the amount of recombinant protein that will be produced. Many common promoters...and steroid regulated promoters commonly used in plant research. Promoter Subtype Promoter Example Activator...promoter is right for me? Here’s a list of some important variables to consider when choosing an inducible...various eukaryotic cell types (mammalian, yeast, plants, etc.) require unique promoters and there is very...’re expressing a gene that may be toxic, it’s important that your inducible promoter not be too leaky....low leakiness. Inducibility level How much do you want to induce transcription of your target gene? The...

...scientists to both quantify Cas9 activity and sgRNA efficiency as well as enrich for mutants through the presence... created CRISPR fluorescent reporter assays to quantify targeted Cas9 cleavage, and repair through homology... in 2017 to enrich and identify desired CRISPR mutants (Wen et al., 2018). SRIRACCHA uses the Piggybac...presence of a fluorescent reporter. Lastly, once a mutant is identified, the PB transposase can easily and...effective gRNA and enrich cells for your CRISPR mutant of interest, give these methods a try.  References...

...diverse libraries of genetic variants. Several methods for generating mutant libraries exist, but none provide...provides a new approach for generating novel genetic variants in bacteria. Read on to learn about the key components...Assisted Continuous Evolution (PACE) method requires constant turbidity of the culture, so bacteria must be ...novel genotypes that result in a spectinomycin resistant phenotype in bacteria. While there are many known... feature which they used to generate bacteria resistant to both spectinomycin and streptomycin, but could...