...is codon usage important? Translation and protein synthesis relies on transfer RNAs (tRNAs). tRNAs bind...rate of translation and thus protein expression. In fact studies have shown that translational efficiency...on an organisms codon usage bias to increase translational efficiency and thus protein expression without...conformation, folding and stability, change post-translational modification sites and even affect protein function...Mauro and Chappell, 2014). Different rates of translation by different tRNAs, including those that exhibit...actually be critical for determining the rate of translation. The ribosome may slow and pause during elongation...optimization may disrupt the fine-tuned timing of translation and ultimately protein function.  In addition...

... Retroviruses are single-stranded RNA viruses that use reverse transcriptase to synthesize complementary...alternative virus, or other strategies may need to be considered if size constraints are an issue. Adenovirus...ample room for most experimental needs, and can transduce (infect) a wide range of cell types with a high... such as a heterologous envelope or a post-transcriptional regulatory element can be used for either type...Adeno-associated virus (AAV) is a small, single-stranded DNA parvovirus. AAV can infect humans and some...replicate episomally, essentially remaining an extrachromosomal part of the host genome that can replicate...response as retroviruses. This virus can also transduce dividing and non-dividing cells, making it conducive...

... pair has focused considerable attention on transcription factors, starting with an established method... relies on antibodies to recognize and bind transcription factors directly, but that’s a slow-going process... it doesn’t work in many cases. In an effort to track down every last one of those gene regulatory proteins...ENCODE project is to look at all 1,500 or 1,600 transcription factors and figure out where they bind to the... ChIP-seq, but it’s really hard for lots of transcription factors. Our groups got together to adapt CRISPR...big difference. We can now look at many more transcription factors. Addgene: How does the system work? ...second plasmid to target an epitope tag onto the transcription factors. By the way, you can actually use this...

... was on the right track. The next insight was to assemble a synthetic transcription activation complex...after natural transcription activation processes. Konermann and her colleagues demonstrated the new system... sgRNAs for each transcription start site in 15,977 human genes. The transcriptional repressor (CRISPRi...each transcription start site in those 15,977 human genes. Weissman and his colleagues demonstrated in ...SAM is an engineered protein complex for the transcriptional activation of endogenous genes and it consists...sgRNA incorporating two MS2 RNA aptamers at the tetraloop and stem-loop 2; 3. The MS2-P65-HSF1 plasmid which...consequences of repressing or inducing individual transcripts. The activator (CRISPRa) sgRNA library uses the...

... its fluorescence emission spectra changes when it goes from a more neutral pH compartment, ­­like the...the mechanisms behind autophagy by tracking what’s being transported to the yeast vacuole (i.e. cytosol...Electron microscopy: Electron microscopy is a traditional method for studying autophagy. This method relies...autophagosomal membranes, so tagging it with GFP allows for tracking the localization or accumulation of pre-autophagosomal...an autophagy-independent manner. Additionally, tracking Atg8p or LC3 doesn’t provide information about...color-emission biosensor named after the brightly-coloured Australian parrot. It’s comprised of two tandem fluorescent...a summary of Rosella’s excitation and emission spectra. DsRed is the permanent fluorescent tag portion...

... and recent advances have allowed scientists to track multiple genomic loci over time using all the colors...CRISPRainbow. Figure 1: CRISPRainbow was used to track the localization of 6 chromosome-specific loci in...labeled dCas9 to a given genomic locus, you can track the localization and movement of this locus in live...depositor Stanley Qi and used in Chen et al. to track telomere dynamics throughout the cell cycle. To ...distances from 2-75 Mbp. Labeling multiple, intrachromosomal loci Building on the work of Chen et al., ...turned their attention to mapping pairs of intrachromosomal loci. The technique successfully resolved ... this work represents the first mapping of intrachromosomal loci, a major benchmark in characterizing ...

...the co-transduction by two viruses would lower editing efficiency, the split editors transduced at levels...colleagues evaluated CreLite for cell tracing using the transgenic zebrafish model, ubi:zebrabow. In this...capacity of 4-5 kb limits the delivery of larger transgenes, such as base editors which have a gene size ...base editor is split into two plasmids and co-transfected into cells for reconstitution. Image from Levy...causes Niemann-Pick disease type C, directly demonstrating the therapeutic potential for the split base... of response and brightness. They also do not spectrally separate from green sensors and blue-light-activated...Image from Beck and Gong, 2019. Broadening the spectral diversity of GEVIs can enable resolving single...

...membrane localization of transmembrane proteins, like rhodopsins, are highly constrained and poorly understood...delivered ChRgers by intravenous injection of PHP.eB, an AAV capsid capable of broad transduction of the nervous... to perform in vivo optogenetic experiments. Intracranial injections of AAV are commonly used to deliver...channelrhodopsins to the brain. This delivery route concentrates lots of channelrhodopsins in one region of the...channelrhodopsin, such as cell localization, kinetics, and spectral properties, must be optimized simultaneously in...channelrhodopsins with desired characteristics. The model was trained to predict if a channelrhodopsin sequence would...neurons and mouse brain slices collected following intracranial injection of an AAV expressing a ChRger. In ...

... GFP scaffolds for transcriptional activation The project began with a frustration many researchers have... to create a complete transcription factor, activating luciferase transcription. Tang et al. coined this...Connie Cepko’s lab allow researchers to activate transcription or Cre recombinase activity only in the presence...suddenly seemed very realistic. Envisioning GFP as a substrate promoting dimerization, Tang et al. tested pairs... in their 293 cell culture system. In cells cotransfected with GFP and the GBPs, GFP linked the two GBPs...this system T-DDOG (Transcriptional Devices Dependent On GFP). In testing the specificity of their system... dsRed, mCherry, and TdTomato do not induce transcription. Thus, T-DDOGs can be used in combination with...

...competent bacterial strain (e.g. DH5α which is dam+). Methylation deficient bacterial strains can be identified...homology-directed repair (HDR) of a CRISPR/Cas9 induced double-stranded break. This site-directed genome editing requires...methylation-dependent endonuclease (i.e. DpnI), and bacteria are transformed with the nuclease-resistant nicked plasmid (the...products of the PCR reaction are pieces of double stranded, circular DNA with staggered nicks, recent data...final PCR products are pieces of linear, double stranded DNA with homologous ends. These homologous ends... In addition, you may want to try different concentrations of template (e.g. 0.1-1.0 ng/μl). Smaller plasmids...to the PCR reaction (usually around 3% final concentration). The DMSO reduces secondary structures of the...

...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...

...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,...

...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...

...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...