...synaptic transmission. Cleavage of VAMP2 renders it inactive, thus inhibiting neurotransmitter release....depositors demonstrated the utility of light-mediated suppression of synaptic transmission in brain slices... proteins scatter sound waves and create ultrasound contrast. At a very low volumetric density and with...vivo using ultrasound in Escherichia coli and Salmonella Typhimurium. This work demonstrates engineered...Plasmids podcast Light-mediated block of synaptic transmission Article contributed by Klaus Wanisch  Listen...enable VAMP2 cleavage, and suppresses neuronal transmission within minutes. Constructs using the photodimerizer...other vital systems in the body. When a microbe travels through a host organism, the expression of some...

...mcherry) are used in high concentrations in combination with low concentrations of vectors expressing the...recombinase. The high concentration of FLEx vectors guarantees that most cells are transfected with all labeling...7624356. PubMed Central PMCID: PMC41457. 8. Schnütgen, Frank et al. 2003. “A Directional Strategy for Monitoring... sites can lead to excision, inversion, and translocation of DNA in a similar fashion as between loxP ...-LDB domains of the fusions allowing Cre/FLP translocate to the nucleus where they exert their recombinase...brain? Cells of a specific brain area can be transfected by in utero electroporation. To achieve sparse... could reduce the plasmids amounts to reduce transfection efficiency. However, if the cellular structure...

...to the strategy adopted, and to the specific reaction you are running. Transformation Transform a few ... them an E. coli  strain known to be sensitive to that antibiotic, and if the strain is growing fine, ... positive clone, try transforming the ligation mix in different bacterial strains (stbl2, NEB stable, ...The PCR could fail to produce a product, the transformation may not result in any cells, or all the colonies... will definitely help in understanding if your strategy will be successful, and to avoid easy mistakes...combination of different ways according to your cloning strategy: Restriction enzyme digestion Be mindful of the...faster cloning. Remember to quantify the DNA concentration of all the samples by gel or by nanodrop to ...

..., the ability to detect mRNA transcripts of toxins and other transcripts of interest is a benefit of this...Microbiome studies have traditionally fallen into studies of who’s there and what are they doing. To ...the organisms present while sequencing of all transcripts gives clues into what the microbes are doing.... scientists introduce an Escherichia coli donor strain into the microbiome to deliver a genetic payload... microbial species. These also contain the RP4 transfer origin as well as a selectable marker and a desired...followed by a reporter gene. The hairpin blocks translation by sequestering the RBS, but binding of the “...microbial community) reveals the RBS and allows translation of the reporter gene resulting in GFP signal....

...PubMed PMID: 25307932. PubMed Central PMCID: PMC4253859. 4. Hart, Traver, et al. "High-Resolution CRISPR...CombiGEM uses iterative one-pot reactions with straightforward restriction digestion and ligation steps to... in each plasmid. Each gRNA combination can be tracked and quantitatively analyzed by sequencing its set...expression of other genetic elements including transcription factors (Cheng et al., 2014) and microRNAs (...including those found at Addgene! Figure 1: Strategy for assembling the barcoded gRNA library pool....before beginning your experiments. Figure 2: Strategy for assembling barcoded combinatorial gRNA libraries...2014): 12462-12467. PubMed PMID: 25114216. PubMed Central PMCID: PMC4151723. 2. Doench, John G., et al. "...

...allowing the four Cre proteins to form a tetramer. A double-strand DNA break occurs within the core spacer...maintained).  Translocation: If the sites are on separate DNA molecules, a translocation event is generated... expression – placing a series of transcriptional and translational termination features with loxP sites...2839833. PubMed Central PMCID: PMC281709. 3. Tissue– and site–specific recombination in transgenic mice. Orban... pun) specific, targeted DNA modifications in transgenic animals, embryonic stem cells, and/or tissue-...recombinase”), is a 38 kDa protein responsible for intra- and inter-molecular recombination at the loxP recognition...spacer of each loxP site and the two strands are ligated, resulting in the reciprocal crossover event. This...

...BioRender.com. Transformation Grade DNA (low quality/low quantity) – Bacterial transformation protocols require...eluting the DNA from the substrate. In a column-based approach, the substrate is a silica matrix fixed... large amounts of high quality DNA. Figure 1 illustrates the three grades of DNA you may need based on...highest-competency cells you will still struggle to get transformants if the plasmid you are using is completely degraded...important reactions like PCR or restriction digests. Transfection Grade DNA (high quality/high quantity) – Depending...Depending on the experiment, mammalian cell transfections can require quite large amounts of DNA (easily...the plasmid DNA from your cleared lysate to a substrate, washing away left-over debris that carried over...

... a strong transcriptional activation domain. Guided by dCas9, VP64 recruits transcriptional machinery ...as two other potent transcriptional activators (p65 and Rta). These transcriptional activators work in ...interest. Upon binding, CRISPRa systems recruit transcription factors to increase gene expression. CRISPRa...CRISPRa methods vary in their transcriptional activators: some methods rely on fusion proteins while others...gene regulation. This can be used to activate transcription during either initiation or elongation, depending...uses specially engineered sgRNAs to increase transcription. This is done through creating a dCas9/VP64 ...at each loci of interest, this allows more transcriptional machinery to be recruited per targeted gene...

...signal downstream of the transcription start site will terminate transcription of most of the ncRNA (Figure... schematic illustration of CRISPR/Cas9-based approaches to study ncRNAs. (A) Transcription start site ... coming, right? Non-coding RNAs (ncRNAs) are transcribed but do not encode proteins. They play important...Using a single guide RNA (sgRNA) to create a double-strand break works well because very small deletions and...sequence can completely disrupt expression of the transcribed ncRNA (Jiang et al. 2014).  Simple knock-out ...lncRNA regulatory elements that are crucial for transcription and downstream function. Rather than attempting...the lncRNA locus, scientists can mutate the transcription start site, splice sites, exons, and/or promoters...

...18930153. PubMed Central PMCID: PMC2644460. 12. Querido, Emmanuelle, and Pascal Chartrand. "Using fluorescent...folded beta-barrel structure, followed by an intramolecular reaction that generates the chromophore (Tsien...and require uptake by the cells. Which of these strategies works best for a specific biological system under...intensity of purified protein at different acid concentrations. Overviews of the pKa values of different fluorescent... tag and several others. Another peptide-based strategy uses an enzyme to covalently label the peptide...specific RNA stem loop-structure (Querido and Chartrand, 2008). Finally, a nuclease-deficient Cas9 can... Since longer wavelengths have better tissue penetration, (infra)red fluorescent proteins are also a better...

...is composed of single stranded DNA, which is particularly well suited for strand invasion, a key step ... are then packaged into AAV virions via transient transfection of 293T cells with helpers, and used to...identical targeting vectors delivered through transfection of naked DNA. Other investigators who had been...inserted downstream of a functional promoter (gene traps). The resultant gene targeting system could be used...the efficiency of homologous integration via gene trap enrichment, whereas pAAV-TK-Acceptor has a conventional...specific frameshift mutations through aberrant double strand break repair, it has proven more challenging to...AAV-based gene editing via the introduction of a double strand break by using a targeted nuclease. Asuri et al...

...found in the extracellular space. The targeted epitopes can be motifs within transmembrane proteins, such...nature, antibodies cannot penetrate the cell membrane; hence, intracellular labeling with antibodies requires...label intranuclear proteins of interest, you can label cytosolic proteins alongside the intranuclear ones...such as receptors, or post-translational modifications on those proteins, like glycosylation patterns....cells. However, there are more techniques and strategies one can employ beyond simply incubating cells...downstream applications, like an in vivo adoptive transfer of those CD8+ T cells, require the cells to be...your sorted cell sample than MACS. An effective strategy to sort out your cells of interest via FACS using...

...Modular fluorescence unit fused with TRansmembrane region-to-IntrAcellular loop linkers” (MTRIA for short),...-gTEMP plasmids here!   Lu K, et al. Intracellular Heat Transfer and Thermal Property Revealed by Kilohertz...new class of sensors for neuropeptides and neurotransmitters. Motivated to develop a biosensor for oxytocin...sensors based on GPCRs recognizing various neurotransmitters, neuropeptides and other ligands - almost ...inserting a circularly-permuted GFP into an intracellular loop of various G-protein-coupled receptors....fluorescent sensor for real-time measurement of extracellular oxytocin dynamics in the brain. Nat Methods....kinetics and localization for imaging synaptic transmission. bioRxiv 2022.02.13.480251. doi: 10.1101/2022.02.13.480251...

...genes from a single transcript, or if you want to prevent read-thru transcription from resulting in the...developing a general translation initiation sequence known as the Species-Independent Translational Sequence (SITS...extensive sequences used for cloning into the final translated products. Negative selection markers built into...3509-3514. Tikhonova, Elena B., et al. "A transcription blocker isolated from a designed repeat protein...of which you can clone your gene of interest. Translation of the standardized leader peptide prevents secondary...encoding your gene of interest from affecting its translation initiation and instead makes initiation heavily...the production of a downstream peptide, the transcription terminators in this series are just the tool...

...techniques used to generate transgenic mouse lines, such as BAC transgenic or traditional knockins/knockouts, ...researchers generating transgenic animals may choose Southern blotting to screen founder transgenic animals for ...fragmented into smaller pieces, run on a gel, and transferred to a membrane for analysis through hybridization...for the inserted transgene because Southern blots have a lower false positive rate than real-time PCR (4...hydroxyl group required for the extension of a DNA strand (8). Adding a small amount of a single radiolabelled...regular nucleotides results in the synthesis of DNA strands of all possible lengths because extension is terminated...prevails. Sequencing whole genomes or profiling transcriptomes (RNAseq) require sequencing of many samples...

...’s lab at Temple University has demonstrated two potential strategies for CRISPR-HIV therapeutics - one...one using dCas9-SAM to activate HIV-1 transcription and destroy infected cells, the other using wild-type... no viral protein. To solve this problem, two strategies have been proposed. The first, “shock and kill...cytotoxicity or the immune response. The second strategy is the simple removal of the HIV-1 genome from...histone deacetylase (HDAC) inhibitors to increase transcription of the integrated HIV-1 genome. For a targeted...dCas9-VP64 fusion commonly used to activate transcription, but with two additional activation domains ...domains (MS2 and p65) to enhance transcriptional activation. Zhang et al. designed multiple gRNAs to target ...

...proteins, which are directly trafficked to cell membranes. Membrane trafficking of farnesylated derivatives... virus post-transcriptional regulatory element (WPRE), commonly used to increase transgene protein levels...has become very useful in lineage tracing; “Zebrabow” was used to trace the development of the corneal epithelium...Brainbow 3.0, 3.1, 3.2 plasmids References: Transgenic strategies for combinatorial expression of fluorescent...2013 May 5. PubMed. Zebrabow: multispectral cell labeling for cell tracing and lineage analysis in zebrafish... also been applied to cell marking and lineage tracing in other systems, such as zebrafish and Drosophila...This system differs from Brainbow-1.0 in that transient Cre recombination is required to prevent constant...

...different DNA strand comes along, it can attack this covalent bond thus joining the two DNA strands and releasing...planning to transform right away OR you can store the reaction at -20C overnight. 4. Transform TOPO Cloning...end cloning. So how does TOPO cloning work? As illustrated in the Figure below, the “A” overhang on the ...DNA sequence 5´-(C/T)CCTT-3' and digests double stranded DNA at this sequence. The energy from this breakage...stored as a covalent bond between the cleaved 3′ DNA strand and a tyrosyl residue of topoisomerase I (1). If...researcher to perform blue-white screening after transformation - self joining of the vector ends results in... minutes will not significantly improve the transformation efficiency).  5. Select and Analyze 10 White...

...of DNA, transcription of DNA to make RNA, translation of RNA to make proteins. That's central to everything...Science describing the development of “transcriptors,” transistor-like digital genetic switches that enable...comprised of well-characterized bacterial transcription and translation initiation elements. Open science with.... We have a strategy that organizes how we start the work, and it begins with the central dogma - replication...reliable gene expression via standard transcription and translation initiation." Nature Methods. 10, 354...cellular computing. These logic gates built from transcriptors are now available at Addgene through the Biobrick... handle on precise, forward engineering of the central dogma at the genome scale, it's going to be tricky...

...contains 11 β-strands and one central ɑ-helix that can be split into 3 parts: (1) β-strands 1-9 (β1-9) and... and the one ɑ-helix, (2) β-strand 10 (β10), and (3) β-strand 11 (β11). Each part contains components ...developed, such as the Forster resonance energy transfer (FRET)-based executioner caspase reporters. In...GFP (GFP1-9 and GFP10-11) by flipping a single β-strand of GFP, hence the name FlipGFP. FlipGFP increases...from β1-9 they will rapidly recombine with β1-9 strands nearby resulting in fluorescence within tens of...11 are antiparallel. To prevent binding, the β strands in FlipGFP were redesigned so that they are parallel...E5 and K5) that dimerize together and force the strands into a parallel conformation. Between β11 and one...