...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...

...mNeonGreen can be used as a fusion tag for a wide range of applications from traditional imaging to FRET...from the coral Clavularia sp. protein CFP484. Hui-wang Ai and colleagues from the University of Alberta...mTFP1 can be used as a FRET donor to mCitrine and mOrange or for live cell imaging of fusions and FRET reporters...is also insensitive to physiologically relevant changes in pH. Find mTFP1 plasmids here! mWasabi: a bright...mutagenesis approach and found that specific amino acid changes favored blue-shifting more than others. During ...particularly with fluorophores that excite in the violet range. For example, mWasabi can be used with Sapphire ...deposit the plasmids at Addgene! References Ai, Hui-wang, et al. "Directed evolution of a monomeric, bright...

...sufficient to prevent any pH change, as we still observed a slight change in pH (from 7.4 to 6.8). It’...tubes (Fig. 1). It’s important to note that the change in pH only occurred when the solution thawed, as...samples were frozen. This was clear by the gradual change in pH visible at the sample interface upon thaw...found that 30 seconds of venting attenuated the pH change caused by storage on dry ice (Fig. 3). However,...CO2 to leave the tubes, and would prevent the pH change upon thaw (Fig.4). To test this, we froze the tubes...stability (Murphy et al., 2013). However, the pH changes in our solutions are transient and return to normal... have no evidence to suggest that the transient change in pH affects AAV function. Furthermore, another...

...body monomers respond to pH changes by undergoing local conformational changes. These tiny movements are... much force can be generated by conformational changes in proteins? How can information propagate through... repeatedly extend and contract in response to changing pH, and they can rupture E. coli spheroplasts ...and retract over many microns in response to pH changes without the need for ATP or other chemical fuel..., R bodies do not exert force by dynamically rearranging into different polymerization states. Instead...vasculature or extracellular matrices) in response to pH changes. Beyond these applications, purified R bodies ...

... other CRISPR plasmids are from the Zhang lab. In fact, the Zhang lab has shared over 40,000 CRISPR-Cas9... different cell types so it is useful in a wide range of applications. This plasmid was the top requested...pSpCas9(BB)-2A-GFP (PX458) - This plasmid from the Zhang lab expresses Cas9 from Streptococcus pyogenes with...generation lentiviral transfer vector also from the Zhang lab is used to deliver Cas9 and your gRNA of interest...genome engineering. For use in human cell lines, the Zhang lab codon-optimized SpCas9. Using the BbsI restriction...BB)-2A-Puro (PX459) V2.0 - This plasmid from the Zhang lab is a mammalian vector that expresses both SpCas9...original publication but its name was subsequently changed to clarify that it does not contain the trademarked...

...into one of those unexplored areas with our color-changing flower, but to do that we’ve had to navigate the...modification (GM) and synthetic biology. The mistrust and anger directed at GM crops has burdened this emerging ...product specifically for consumer sales. Flowers that change color throughout the day cannot be developed through...of basic research standing behind it. Our color changing flower involves two basic components: the pigments...combine these components to develop flowers that change color. Without this basic research – this comprehensive...sometimes have limited distribution. We hope that will change with Addgene’s expansion into plant synthetic biology... GreenGate, and we are excited to see how this exchange of genetic tools facilitates plant science. Challenge...

...29581308. PubMed Central PMCID: PMC5899461.  Zhang, Liangyu, et al. "The auxin-inducible degradation (AID...and annotations extracted from other databases (Mangone et al., 2008).  OpenWorm OpenWorm is an open source...conditionally deplete proteins of interest in C. elegans (Zhang et al., 2015). In this system a modified TIR-1 protein...sequences upstream of genes to stimulate expression (Wang et al., 2016). In 2018, the lab expanded on this...covalently ligate their flanking peptides together (Wang et al., 2018).” With the split system, only cells...PMID: 23172286. PubMed Central PMCID: PMC3531189. Mangone, Marco, et al. "UTRome. org: a platform for 3′ ...119 + selection." MicroPublication 1 (2018): 2–3. Wang, Han, et al. "cGAL, a temperature-robust GAL4–UAS...

...donor-acceptor pair and a change in FRET efficiency correlates with a change in the distance and/or orientation...fluorescent protein (i.e. rearranging the start and end positions without changing the order of the amino...to a change in the environment which alters the FRET efficiency. Detecting certain cellular changes with...biological processes occur within the typical FRET range, FRET efficiency is used to infer an interaction... protein based FRET pairs with improved dynamic range for fluorescence lifetime measurements." PloS one...studying protein-protein interactions by measuring the change in FRET efficiency, from which conclusions regarding...

...your expected range. For example, calcium in the ER lumen reaches the sub-millimolar range, much higher...calcium, (2) allosteric rearrangements of the sensor, and (3) subsequent change in fluorescence only occurs...systems tend to have slower, but larger, calcium changes. The GCaMP6 family of sensors are commonly used...compartments can vary greatly. Additionally, the calcium changes you will observe in your experiment could be subtle...are available to simultaneously measure calcium changes in the ER, cytosol and mitochondria), the Cameleon-based...ratiometric GECO sensor (GEX), where one wavelength changes with Ca while another wavelength is insensitive...sensors have also been re-engineered to measure changes in calcium specific to the neuronal cell body or...

... positive change in the scientific enterprise and the way it is funded and to learn tangible skills necessary...necessary for affecting change. On Friday, the symposium includes a panel discussion and keynote by former...the Advocating for Science Symposium Boston 2016 Changes since the Future of Research Symposia Ongoing ...initiated. Recent developments promise to further change the landscape facing all scientists, both young...available avenue. Interestingly, both of these changes share a common thread that has the potential to...for the future No matter your opinion on these changes, it is likely that the shifts caused to the practical...example, implementation and compliance for the FLSA changes, including finding money in already tight lab budgets...

...T7E1, and Sanger sequencing. However, recent studies suggest that both Surveyor™ and Sanger may not be...et al. 2013, Vouillot et al. 2015). Meanwhile, Sanger sequencing is laborious, time consuming and cannot...heterogenous populations (Bell et al. 2014). Further, Sanger sequencing has a lower detection limit of 50-20%...Inexpensive, simple Low-throughput, low sensitivity Sanger Sequencing Provided 20% (variable) Sequence-level...IDLV for in vivo detection (Gabriel et al. 2011, Wang et al. 2015, Osborn et al. 2016) and HTGTS (Frock...magazine.scienceforthepeople.org/.     References 1. Fu, Yanfang, et al. "High-frequency off-target mutagenesis ...., et al. "Improving the limit of detection for Sanger sequencing: a comparison of methodologies for KRAS...

...things off, “We are all capable of contributing to change.”  Check out Joanne's Reddit AMA The keynote speaker...spoke eloquently and vehemently about the need for change.  Dr. Bourne strongly believes this is an amazing...PI driven basic research, there is resistance to change, public support is weak, government support is ...Finally, the science publishing system needs to be changed.  Dr. Bourne charged the audience to use their ...engaged in training excellence to begin effecting change.  Panelists call for action Each of the speakers...of how you don’t have to be in academia to make change.  He talked about his own unique way of funding... in the “best” journals.  He pushed for drastic change in the way we train scientists to start addressing...

...introduction of specific sequence changes. To generate a specific sequence change the cell must undergo homology-directed...extensively for the purpose of making sequence-specific changes in human induced pluripotent stem cells (iPSCs)... (ssODN), which contains the intended sequence changes to be incorporated into the edited genome. In mammalian...CRISPR/Cas9 blocking mutations in either the PAM (changing one of the G’s to an A,T or C – but avoid NAG)... to be silent. In our view, it’s always best to change the PAM site, however we have also demonstrated...mismatch in the binding sequence, we recommend changing several bases as close to the PAM as possible ...blocking mutation together with the intended sequence change and screen a few hundred clones to find the right...

... Yuan S, Wang Y, Hu B, Yip CC-Y, Tsang JO-L, Huang X, Chai Y, Yang D, Hou Y, Chik KK-H, Zhang X, Fung ...Guo L, Guo R, Chen T, Hu J, Xiang Z, Mu Z, Chen X, Chen J, Hu K, Jin Q, Wang J, Qian Z (2020) Characterization...JD, Chu AW-H, Zhou J, Lung DC, Kok K-H, To KK-W, Tsang OT-Y, Chan K-H, Yuen K-Y (2020) Comparative tropism...

... world over. Scientists have traditionally used Sanger sequencing to validate these constructs, as the...relatively low cost and quick turn-around time of Sanger sequencing have historically matched the needs ...and accurate sequencing (“reading”) of DNA, have changed our collective thinking about the feasible size...each sample remain highly normalized across a wide range of input DNA concentrations. This process is explained...plasmids on a plate) will typically have a wide range of input quantities. This variation is problematic...produced from large numbers of samples with a wide range of input concentrations (via proprietary chemistry...differences in input concentration across a 100-fold range (1ng – 100ng) are normalized to two-fold variation...

...month project to validate some genomic changes, so this arrangement was ideal for everyone.  The cutting...fit for me, then I didn’t need to explain why I changed jobs. I highly recommend taking on a similar position...major factor held me back; I do not have a second language. During the recession there were many qualified...qualified scientists with experience and a second language in Spanish or French, and so they were ideal candidates...advantage of the fact that science is a global language that allows you to work anywhere and build a network...this company for several years, I was ready for a change.  Working abroad: Research in Australia I then ... fit in with the company and that my ideas and changes were welcomed, which is a benefit over larger companies...

...deaminase. Base changes from C•G-to-T•A are mediated by cytosine base editors (CBEs) and base changes from A•T-to-G...capabilities. Base editors make specific DNA base changes and consist of a catalytically impaired Cas protein...target site, and the deaminase induces the DNA base change without a DNA double-strand break. But base editors...is that the editing domain must induce the base change before the Cas domain lets go of the DNA. ABEs ...hold on” long enough to the DNA to induce a base change. Because of this, ABEs are only weakly compatible... of the fetal hemoglobin gene, ABE8e made both changes simultaneously with 54% efficiency, about 7 times... DNA base swaps, so called point mutations that change C to T, G to A, A to G, and T to C, represent about...

...biochemical activity Article contributed by Angela Holmes Jin Zhang’s lab is developing new super-resolution...conformational change that placesTagRFP-T and Dronpa in close proximity. By monitoring changes in TagRFP-T... to blue light, CRY2 undergoes a conformational change that permits it to bind to the CIB1 (cryptochrome-interacting...optogenetic system (OptoSTIM1), caused two-fold faster changes in intracellular Ca2+ levels than the original ...Synthetic decaffeination operon Article contributed by Angela Kaczmarczyk  Listen to the synthetic decaffeination...techniques for live-cell imaging of the dynamic changes in biochemical activity within the cell. Their ...

...References Dong D, Guo M, Wang S, Zhu Y, Wang S, Xiong Z, Yang J, Xu Z, Huang Z (2017) Structural basis... 2017 and last updated Jul 23, 2020 by Jennifer Tsang. CRISPR-Cas technology is constantly evolving. Variants...10.1016/j.cell.2017.07.037 Li C, Psatha N, Gil S, Wang H, Papayannopoulou T, Lieber A (2018) HDAd5/35++...//doi.org/10.3390/v10120695 Pawluk A, Amrani N, Zhang Y, Garcia B, Hidalgo-Reyes Y, Lee J, Edraki A, Shah...https://doi.org/10.1016/j.cell.2016.11.017 Shin J, Jiang F, Liu J-J, Bray NL, Rauch BJ, Baik SH, Nogales ...