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...nicking of the unedited strand and encourage the cell to use the edited strand as a template during DNA...on a fusion enzyme of Cas9 nickase + reverse transcriptase and a prime editing guide RNA (pegRNA). The ...) complementary to the genome and a reverse transcriptase (RT) template including the desired edit. ...by the type of edit, genomic context, delivery strategy, cell type, and goal of the experiment. Now, onto...., 2019). Test different lengths of reverse transcriptase template, starting with about 10–16 nucleotides...pegRNA primer binding site (PBS) and reverse transcriptase template (RTT). Created with BioRender.com. ...from re-binding and nicking the newly synthesized strand before heteroduplex resolution, which can lead ...

...bacterial transformation One protocol that has been automated is high throughput transformation. With our...platforms that fit 48 well q- trays allowing us to do up to 96 transformations in one run compared to doing... be useful if you’re transferring samples from 96 wells plates onto agar q-trays where the samples must...easier and more efficient. Here, I’ll discuss transformation, DNA purification, and filling vials with agar...deposits (over 100 deposits per month!), manual transformations were taking a lot of time. Enter the Biomek... We wanted a machine that would improve our transformation protocol and, after looking at a few options...doing about 50 transformations in the same amount of time by hand! Setting up a basic protocol on the...

...their mutations corrected, and the T-cells were transferred back into the infants. Almost all patients have...repertoire (1). In this example, it was possible to transduce the T-cells outside the body and thereby correct...techniques may hold the key. In the simplest form, translating gene therapies from conceptual design to clinical...deliver it and identifying a suitable route of administration. However, until recently safe delivery of nucleic...that carries a small, approximately 4.7 kb single stranded genome. AAV belongs to the family Parvoviridae...disease-associated mutations. AAV vectors maintain persistent transgene expression over many years in postmitotic, long-lived...2008. Following a single dose of subretinal administration of rAAV2 carrying the human gene RPE65, patients...

...and called for the fast-tracked development of diagnostics. Mostly transmitted by Aedes mosquitoes (aegypti...international travel, the Zika virus is expected to expand into heavily populated regions of South, Central, and...between global strains of the Zika virus (Asian-, African- and American-lineages). Strain discrimination...room temperature (Pardee et al, 2014). As a demonstration of the diagnostic potential of these combined... for the detection of antibiotic resistance and strain-specific diagnostics for the Ebola virus. Our original...and electronic companion technologies that has transformed the assay’s capabilities. Specifically this includes... now detects samples at clinically relevant concentrations (1-3 fM; 1x106-fold lower than before), and...

...electromagnetic spectrum – the spectral region in which mammalian tissues are nearly transparent. As Verkhusha explained...photoacoustic contrast agents for two-color imaging in animals. The approach relies on ultrasound waves as ...tumor. They wanted to find something better. Transparency window That’s exactly what they reported doing...explained it to me, the transparency window of mammalian tissues is set by the properties of hemoglobin ...corals, but all of them fluoresce outside the transparency window of mammalian tissues,” Verkhusha said...near-infrared fluorescent proteins within this transparency window, so now, we could see deeper.” Adding...Communications last year, “The capability to control spectral properties of PA FPs with light of specific wavelength...

...nickase. Nickases create a single-strand rather than a double-strand break, and when used with two adjacent...the target strand. Conversely, the H840A mutation in the HNH domain creates a non-target strand-cleaving... how IDT (Integrated DNA Technologies) first demonstrated how CRISPR nickases improve homology directed...strand-cleaving nickase. Instead of cutting both strands bluntly with WT Cas9 and one gRNA, you can create a staggered...to each other? The gRNAs must target different strands to create a DSB, but this can be accomplished with...experiment to insert an EcoRI site using a single-stranded oligonucleotide (ssODN) donor. Using a D10A nickase...across the 51 nt region using either a top or bottom strand ssODN donor with 40 bp homology arms. The HDR efficiency...

...better. More transcription factor binding sites in a promoter lead to higher transcriptional activation....Fluorescent protein fusions One of the easiest ways to track a given protein is to fuse it to a fluorescent protein... present when the protein is at a very high concentration. Overexpressed proteins also have the potential...fluorescent signal and enables single molecule tracking  within living cells without affecting protein...thereby creating a single-molecule reporter of intracellular processes. Initially, Tanenbaum et al. observed...molecules very effectively - they even managed to track run lengths of the motor protein kinesin across ...cell physiology. Having seen that mitochondrial tracker GFP-mitoNEET can impair mitochondrial function,...

...number of transformants); the greater the number of inserts, the lower the number of transformants. To compensate...containing the transformants onto the selection plate. Figure 2 shows representative transformation plates obtained...cassette, and illustrates how the volume of the 1 ml outgrowth spread on each transformation plate can be...prefers ligation of Watson-Crick base pair substrates, it demonstrates significant activity on some mismatch-containing...the number of transformants while maintaining high levels of correct assemblies) translate to faster assembly...experimentally-derived DNA Ligase fidelity data. Our work demonstrates that it is now possible to achieve 20+ fragment...background by antibiotic selection. Screening of transformants by colony PCR confirmed the insertion of the...

...character traits and skills that you hope to grow.  Find mentors who take their responsibility to train you ...decisions without much logic - even choosing a training lab where we will spend the next 6 years. Know...about this lab’s reputation? If you are choosing a training lab, I strongly suggest you complete a work rotation... also be a sign that the lab head has trouble attracting scientists. To get a better idea of why the lab... head is an absentee advisor. They are always traveling, or too busy to interact regularly with the newer...happened to her a year later. I hear too many science trainees get warnings and still think “but that won’t happen...follow the academic career path taken by their training lab mentors. ~80% of you will be in jobs outside...

...state machines in living cells” (1), into a more straight-forward, concise explanation of how to adapt our...between them (Fig. 1A below). In the absence of extra recombinase-specific co-factors (which should be... what order they were added. Figure 1D below demonstrates an example of a register that enters distinct...might one read the state of an RSM? The most straightforward way to read the state of an RSM is to sequence...sent the PCR product for sequencing.  A less straightforward, but more population-wide (as opposed to single-cell...our paper. In our opinion, the most convenient strategy is the fluorescent reporter based method since...promoters: the BxbI gene downstream of the anhydrotetracycline (ATc)-inducible promoter (PLtetO), the TP901...

...ETH Zürich. “Instead of a graphical abstract, it’s a musical abstract,” says Di Ventura, a professor at ...from one that represses transcription to one that activates transcription.However, this system lacks spatio-temporal...create these images, the scientists used a strain transformed with pBLADE expressing superfolder GFP instead...barbara_synbio) May 13, 2021 The original AraC transcriptional regulator Their system is based on the AraC...AraC protein that controls transcription from the PBAD promoter. “AraC is widely used in synthetic biology...It relies on the sugar arabinose to activate transcription by causing conformation changes in the AraC ...domains of the BLADE dimerize and activates transcription. The team created two constructs, among others...

...(single vs. double stranded) all depends on the size of the knock-in. Single strand oligonucleotide donors...Taking the road less traveled and generating a knock-in cell line instead of a knock-out? We’ve got you...knock-out. The goal of a knock-out is to disrupt DNA translation by generating a frameshift mutation. The majority...pathway cells already have for repairing DNA double strand breaks – (HR). HR is the dominant homology-directed... Fig. 1: Early steps in repair of a DNA double strand break by homologous recombination.    Designing...synthesis limits of oligonucleotides. These donors traditionally have larger homology arms in the 500 bp to 1... 1 kb range, however, shorter homology tracts have recently been used with some success (Yu et al., Nat...

... CRISPR over traditional antibiotics The potential advantages of CRISPR over traditional antibiotics are... years after introduction. Current antibiotic strategies are nonspecific - they harm any bacterial cell...beneficial flora. Currently, there is no antibiotic strategy available to target only virulent or antibiotic... joining machinery, so a CRISPR-induced double-stranded break (DSB) in the genome is lethal. If this DSB...treatment were sufficient to wipe out the virulent strain. Bacteria not killed by the first round of treatment...just 24 hours.   Figure 1: Single-phage strategies for bacterial CRISPR targeting. Phage-derived...would be needed for an in vivo therapy. Another strategy for CRISPR antimicrobials combines them with both...

... the absorbance/transmission of light through a liquid to determine the concentration of substances in...ladder with known concentrations. Many commercial DNA ladders will tell you the concentration of each band ...spectrophotometer measures these absorbances using UV-transparent cuvettes. First you measure the absorbance of...absorbance measures give you an idea of the concentration of your DNA prep and whether there are any other...the A260 reading, you can calculate the DNA concentration. Generally, A260 of 1.0 is equivalent to 50 ...the following formula to estimate your DNA: Concentration (ug/ml) = A260 reading x dilution factor x 50...However, there’s limited sensitivity at low concentrations of DNA and it can’t distinguish between DNA...

...kinetics which are illustrated in figures 2 and 3.  Figure 2: Glutamate titration curves for the ... glutamate neurotransmission.” Nature Methods (2013) PubMed PMID: 23314171. PubMed Central PMCID: PMC4469972...accurately differentiating between synaptic vs. extrasynaptic glutamate release. Building on the previous ..., GPI, and SGZ, to improve neuronal membrane trafficking and localization. Improved photophysical properties... Figure 1: 1P and 2P excitation and emission spectra of SF-Venus-iGluSnFR.A184V, iGluSnFR3 v82, and ...Supplementary Figure 3.   Figure 3: pH titration curves and ∆F/F0 for the different iGluSnFR variants...al…, Supplementary Figure 2.   Improved trafficking and localization through different C-terminal...

...methanol from the media. This feature makes the strain tractable for peroxisomal studies on biogenesis, degradation...affordable, and it grows fast to boot. The yeast strains S. cerevisiae and S. pombe have dominated the research... blog we will review an alternative model yeast strain and discuss why you may want to consider a yeast... between two of the most frequently used yeast strains: S. pombe (fission) and S. cerevisiae (budding)... to its fermentation abilities and other useful traits. The “biotech yeast” You’re probably wondering...differences between K. phaffii and other yeast strains.     S. cerevisiae S. pombe K. phaffii...and bears more similarity to the ancient yeast strains and metazoan cells.   Applications for K. phaffii...

... system, however, RepZ concentration is controlled at the level of translation. RepZ mRNA must fold into...pseudoknot” held together by intramolecular base pairs for the protein to be translated. An antisense RNA called...the proteins cooperatively can separate the DNA strands. Created with BioRender.com. Once DnaA breaks...it recruits helicases to continue unwinding the strands. This creates two replication forks that move in...elongation phase, a DNA polymerase synthesizes a new DNA strand at each replication fork. Termination sequences...act as a feedback mechanism to reduce further transcription. Replication is also regulated by the balance...newly synthesized DNA, blocking binding sites, or trading out ADP for ATP to re-activate DnaA. These regulatory...

...use them in, is known as your gating strategy.  Gating Strategies   The series of markers you use to define...isolated.  Following a gating strategy Figure 4 shows a gating strategy from a published experiment (Barlow-Anacker... define your population is called your “gating strategy.” This series of markers are set up sequentially...(Barlow-Anacker et al., 2017). This gating strategy is used to identify two types of dendritic cells (...to each other.        Figure 4: Gating strategy used in Barlow-Anacker et al., 2017 to identify...populations on a plot.      Figure 5: The gating strategy from Barlow-Anacker et al., 2017, with quadrants... lot longer than learning how to read a gating strategy.  Other information FACS plots often contain ...

...tRNAs contain internal transcriptional elements that enhance pol III transcription, potentially increasing...protoplasts. Compared to matching traditional gRNA-expressing constructs, transcript levels of PTG1 and PTG2 were...various tRNA expression strategies? No worries, I've summarized the three strategies described in this post...viruses have illustrated the power of polycistronic gene organization, and its translation to CRISPR/Cas9...lower genome editing efficiency. One previous strategy for multiplexing gRNAs used the RNA nuclease Csy4...base Csy4 recognition sites from a polycistronic transcript. Although this method shortens construct size...coexpression of Csy4 with Cas9 and the polycistronic transcript. As nucleases may have off-target effects and...

...SLC6A8 mutations in Creatine Transporter Deficiency Creatine Transporter Deficiency (CTD) is a rare X-... to be transported from the blood and cross the blood brain barrier using creatine transporters. However...However, mutations in the transporter SLC6A8 impede this transport and result in global creatine deficiency... the SLC6A8 gene, also known as the creatine transporter. The Association for Creatine Deficiencies (ACD...metabolic demand that is powered by ATP. A molecule central to the regeneration of ATP from ADP is creatine...the lab observed abnormal cell migration and contraction. The lab hypothesized that this is abnormally...identify whether any rescue cell migration and contraction defects. The lab also plans to use CRISPR plasmids...