...large changes in binding affinity with light stimulation. Indeed oLID showed only a two-fold change in affinity...variant of PSmOrange, a photoswitchable fluorescent protein which transitions from emitting orange (λ= 565nm...response regulator. These changes reduced system leakiness and increased the dynamic range, resulting in more... and a mostly c-terminal nuclease domain. Feng Zhang's group utilized the bi-lobed architecture of Cas9...constructs or as the fully assembled pMemoryArray.   Yang et al., Nat Methods 2014 Dec;11(12):1261-6. doi:...order to determine the relationships between pH changes and cellular events. To overcome the issues with... of varying fusion protein expression levels. Zhang et al., PLoS One. 2012;7(8):e43072. doi: 10.1371...

... consists of NLuc fused to the more penetrative orange fluorescent protein fusion (OgNLuc). Both fluorescent...for excitation. The result was the cyan-excitable orange fluorescent protein, CyOFP1. CyOFP1 was molecularly...and improved maturation time compared to similar orange fluorescent proteins. In their work, the authors...great imaging tools generate cyan, yellow, green, orange and red fluorescence. The paper reports a wide ...Plasmid #85199), Yellow (YeNL; Plasmid #85201), Orange (OeNL; plasmid #85202) and Red (ReNL; plasmid #85203...When the responsive protein binds to calcium it changes conformation and brings the two fluorescent proteins...sought to alleviate many of these challenges by exchanging the fluorescent donor for a bioluminescent donor...

...identify how MagLOV’s structure changes under magnetic field. Such changes could be harnessed or enhanced...proteins. Motivated by curiosity Although a wide range of fluorescent proteins and optogenetic tools exist... good ol’ EGFP — showed a slight but detectable change in fluorescence when a small magnet was passed ...Fluorescence intensity of EGFP-FlavinTag in E. coli changes in response to a ~25 mT magnet. Gray regions indicate...could be expressed and purified. Although a ~1% change in signal might not make a very powerful tool, ... Q513K, G528K, D540M) that shows a fluorescence change of ~75% in response to the magnetic field (Figure...MagLOV-based tools, just as light-induced conformational changes are the basis of many optogenetic tools. And, since...

...and Types of Edits with Low-Cost Sanger Sequencing   ICE uses Sanger sequencing data to produce quantitative...tools. For example, we examined ICE analysis of Sanger sequencing data alongside the analysis of NGS-based...sequencing. To use the tool, you start by uploading your Sanger sequencing files, either one at a time or as a ...targeted gene.   Analyze Complex Edits   Traditional Sanger sequencing-based analysis tools are unable to detect...unedited (control) populations is PCR-amplified and Sanger sequenced. ICE compares these sequence traces to...software tool is quick and easy. Simply upload your Sanger sequencing files and provide basic information ...file. On the plot, the green (edited sample) and orange (control sample) lines should be close together...

...medium. Daily medium exchanges help alleviate cellular stresses associated with pH changes and resource consumption... ion channels, and growth factors resulting in changes to the cell line’s growth and behavior. Signs of...In addition to benefitting your cells, medium exchanges may also impact the quality of the lentiviral ...harvest. Studies have demonstrated that extreme changes in osmotic pressure and pH can destabilize the ... receptor to infect cells and have a broad host range. However, viruses pseudotyped with other envelope... To overcome this issue, try transducing with a range of dilutions of your lentiviral prep. Hopefully,...conditions for specific applications, stick with them! Changes in such variables as cell density, volume, incubation...

...proteins change configurations or binding partners, the epitopes available for binding change as well....often change shapes and epitope accessibility naturally, by binding to other proteins, changing locations...well. If this change hides the epitope that the antibodies used to recognize the protein, your antibody will... you're working with antibodies.) Conformation changes usually fall into one of two categories: ones induced...  First, let’s think about induced conformation changes. Several types of assays, such as ELISAs and Westerns... some of the bonds keeping the protein folded, changing it from its native state to a less folded, or ...the available epitopes in any given protein might change depending on the conditions and the assay can help...

...dynamic range of your FRET assay (Figure 2). These labeling methods are freely interchangeable and new... Central PMCID: PMC26749. 9. Ni, Qiang, Denis V. Titov, and Jin Zhang. "Analyzing protein kinase dynamics...1999), monitoring kinase activity (Ni, Titov, & Zhang, 2006), quantifying antibody/antigen interactions...Saraheimo et al., 2013), and visualizing structural changes in the ribosome during protein translation (Ermolenko...protein mass into your protein and so structural changes due to this bulky insertion are inevitable. Most... a problem, as long as they are beyond the FRET range of the donor (i.e. >100 Å). Researchers have developed... above can be paired together to measure a wide range of molecular distances (see Table). However, proper...

... Habib, N., Hsu, P. D., Wu, X., Jiang, W., Marraffini, L. A., & Zhang, F. (2013). Multiplex genome engineering...complex (blue subunits) with a guide RNA (green and orange) bound to the target DNA with Cas3 (pink). Complex...double-stranded breaks caused by Cas12 create short 3’ overhangs, which leads to slightly increased efficiency ...., Lander, E. S., Voytas, D. F., Ting, A. Y., & Zhang, F. (2017). RNA targeting with CRISPR–Cas13. Nature...Makarova, K. S., Macrae, R. K., Koonin, E. V., & Zhang, F. (2023). Uncovering the functional diversity ... O., Franklin, B., Kellner, M. J., Joung, J., & Zhang, F. (2017). RNA editing with CRISPR-Cas13. Science...., Zhou, X., Hou, Z., Lozen, M. L., Nam, K. H., Zhang, Y., & Ke, A. (2024). Exploiting activation and ...

...bacteria Article contributed by Will Arnold Baojun Wang’s lab recently developed a CRISPR tool for the specific...called the upstream activating sequence (UAS). The Wang Lab created a tripartite system consisting of dCas9...other bEBP/UAS pairs, and displays high dynamic range. However, the promoter in question must be σ54-dependent... Genetically encoded voltage indicators (GEVIs) change fluorescence in real time when neurons or muscle...technical difficulties such as small fluorescence changes or a requirement for very high excitation light...to work with.  The Gottschalk lab has surveyed a range of microbial rhodopsin GEVIs in excitable cells ... that can be used to monitor real time voltage changes in worms and have made these constructs available...

...transcriptional changes. Using this road map, and what was known about previously characterized TFs, the Zhang group...interest to the scientific community. Recently, the Zhang lab generated a TF library (aka the MORF Collection...unique isoforms transcribed from these sites. The Zhang lab generated a comprehensive library of each unique...human TF can contribute to differentiation, the Zhang lab transduced human embryonic stem cells with the... is a useful finding. Transcriptional program changes from this screen successfully differentiated cells...each TF help streamline identification. While the Zhang lab controlled their multiplicity of infection so... transformation and amplification, refer to the Zhang lab’s previously published protocol and its relevant...

...reality – a government cannot declare that gravity change direction and make things fly up when we drop them...outcomes are actually possible for a given policy. In exchange, the government protects (and often funds [3])...scientists has been rising as evidence of climate change, the safety of genetically modified organisms (...90% of active research scientists believe climate change is mostly due to human activity while much less...administration’s actions, including taking down the climate change page on whitehouse.gov, freezing grants, and proposing... Agency, and the National Institutes of Health endanger scientists’ ability to uphold their side of the...advocating for science education and literacy that this changes.  2) Lumping all scientists (and consequently their...

... control sample. Southern blotting applications range from confirming the results of cloning or knockout...days of development. Today, researchers use both Sanger and next-generation sequencing techniques to answer...see below). Importantly, although their role has changed, restriction enzymes are certainly not dead yet...next-generation sequencing techniques Introduced in 1977, Sanger sequencing, also known as chain-terminating or ...the order of nucleotides in a string of DNA (7). Sanger sequencing uses modified nucleotides, called dideoxynucleotides... and accessibility led to the widespread use of Sanger sequencing. Building upon this technology, radiolabelled... sequencing techniques do not typically rely on Sanger sequencing but instead use methods like pyrosequencing...

...and Screening via Transcriptional Output-TANGO (PRESTO-TANGO) to screen the NCC-1 library of approved .... Cell Rep. 2015. PubMed PMID: 26190106 PRESTO-TANGO: An open-source resource for drug discovery Nearly.... Bryan Roth's lab has a newly available PRESTO-TANGO kit which allows researchers to interrogate the ...publication, Kroeze et al. describe their enhanced TANGO arrestin recruitment assay incorporating 314 codon-optimized...cell line expression. In a twist on the classical TANGO assay, Kroeze et al. developed a new approach termed...discovery. Researchers can use plasmids from the PRESTO-TANGO kit directly for drug screening or easily shuttle... more rapid screening by restriction digest and Sanger sequencing, respectively. The speed and simplicity...

...outside of their natural environment. Thus, the Harris Wang lab at Columbia University developed a method for...both Gram-positive and Gram-negative cells.  The Wang lab further developed a modular suite of plasmids...replicative origins that can be used for broad or narrow range of microbial species. These also contain the RP4... RSF1010 origin, an IncQ origin with broad host-range (Meyer, 2009). The vector retains the oriT for delivery... by conjugation. The labs screened for the host-range of the plasmids and suitability of various antibiotic...designed to study the bee microbiome, these broad-host-range plasmid could also be used to study other microbiome... the toolkit for modular assembly of broad host range plasmids Counting cell division in the gut microbiota...

...register and catalyzes their recombination, thereby changing the state of the RSM. If an attB-attP pair is ...orthogonal attB-attP pairs per recombinase may be arranged on a register (Fig. 1C below). If attB-attP pairs...approach is to interrogate state with qPCR. The re-arrangement of DNA segments in each state of a register enables...methods of state interrogation mentioned above (Sanger sequencing, qPCR, and fluorescent reporter-based...each come with their advantages and disadvantages. Sanger sequencing is simple and the most accurate method...directly reads the state of a register. However, each Sanger sequencing reaction can only be performed on one...or if it requires reading so many registers that Sanger sequencing would be too expensive, then the qPCR...

...Wei Y, Liu F, Ying W, Zhang J, Tang C, Zhang X, Xu H, Shi L, Cheng L, Huang P, Yang H (2018) In vivo simultaneous...experiment! References Bikard D, Jiang W, Samai P, Hochschild A, Zhang F, Marraffini LA (2013) Programmable...for each loci, CRISPR/Cas systems only require changing the programmable guide RNA. Gene activation by... histone acetyltransferase domain capable of rearranging chromatin structure. This method has shown higher... Habib N, Gootenberg JS, Nishimasu H, Nureki O, Zhang F (2014) Genome-scale transcriptional activation...

...product no one has ever seen before (flowers that change color throughout the day) and doing this with very...our lives and took this daring risk? What moment changed our whole outlook on the possibilities in life ...published a paper a year (or less) and limited the exchange of information to the public and to other academic...fair game for speculation - in fact, our color changing flower project was inspired by a Facebook comment...public is can be fearful of this technology and angry at the agriculture companies that produce GMO food...the detector plants, we expected to see that same anger. Instead, social media comments were more often ...we hit upon the idea of building a flower that changed color throughout the day. It would be a beautiful...

...chromoproteins are just now being developed for a broad range of biological inquiries.  What is a chromoprotein... plasmids (meffRed, eforRed, asPink, spisPink, scOrange, fwYellow, amilGFP, amajLime, cjBlue, meffBlue...specific purposes.     Chromoproteins for allelic exchange Figure 3: pTOX plasmids expressing amilCP... create negative selection markers for allelic exchange. These vectors contain a toxin gene (regulated...rhamnose-inducible promoter) and a chromoprotein gene. Allelic exchange occurs through two sequential homologous recombination... the tsPurple chromoprotein.  Find the allelic exchange vectors here! ShadowR, a novel FLIM-FRET acceptor...Image: Murakoshi et al., 2019. ShadowR is an orange light-absorbing chromoprotein that Hideji Murakoshi...

...s clear that CRISPR-Cas9 technology has really changed the game for anyone looking to quickly and easily...screening. They and their colleagues including Feng Zhang used the GeCKO library to identify genes essential...lentiviral CRISPR libraries developed by MIT's Tim Wang and colleagues, including David Sabatini and Eric...limited to modification of a few loci at a time,” Wang said. “It was very difficult to screen in a pooled...targets at once. That’s what our plasmids allow.” Wang’s library is designed to target a smaller set of ...titer due to the smaller size of the plasmids. The Zhang lab, on the other hand, uses a 1 plasmid, single...target any sequence in the genome. Tips and tricks Wang offers this tip: “It's quite important, at every...

...fused to the N-terminus of LSSmOrange pLSSmOrange-C1 Orange Mammalian Express a gene of interest fused...GFP variant) commonly used with mRuby2 pLSSmOrange-N1 Orange Mammalian Express a gene of interest fused... (the acceptor) by means of intermolecular long-range dipole-dipole coupling." The emission of the acceptor...FRET has also been used to measure conformational changes, cleavage activity using FRET-based biosensors,...fused to the C-terminus of LSSmOrange mRuby2 Red Mammalian Expresses mRuby2 (a RFP variant) commonly used...method useful for dynamic experiments in which FRET changes are large. Two other methods are commonly used... such as pH or autofluorescence background, can change the fluorescence decay time and have to be taken...