...per cell increases 4-5 fold (for pUC plasmids) or ~2 fold (for p15a, R6K, and ColE2 plasmids) as cell division...biotechnology 36.4 (2018): 352. PubMed PMID: 29553576. 2. Kittleson, Joshua T., Sherine Cheung, and JChristopher...rounds of replication are initiated before the cell has divided. This means that genes located near the...problems. Rapidly dividing cells initiate genome replication more than once per cell division and thus, genes...genes closer to the origin of replication will be overrepresented compared to those further away. As a...maximal gene expression near genomic origins of replication, the stabilized promoters created by Segall-Shapiro...the control of IPTG. Because RepA is a plasmid replication initiator protein, they could alter copy number...

...be decorated with proteins and remain functional, 2) break membranes outside of their natural context ...PubMed PMID: 3759909. PubMed Central PMCID: PMC213417. 2. Schrallhammer, M. et al. Tracing the role of R-bodies... modified to extend at different pH levels. Domesticating R bodies We have shown that all three of these...actuators that could be used in a variety of applications. For example, you could use them to lyse cells...matrices) in response to pH changes. Beyond these applications, purified R bodies could also perform as actuators...PubMed PMID: 26814170. Find Plasmids from This Publication at Addgene 4. Petroni, G., Spring, S., Schleifer...

... for targeted removal of H3K4me1/2 and H3K9me2 histone modifications. Like the TALE-LSD1 system described...Discovery 2: . https://doi.org/10.1038/celldisc.2016.9 Find plasmids from this publication at Addgene...form or as ready-to-use lentivirus. Figure 2: dCas9-Tet1 demethylates cytosines at promoters and...epigenome (the sum of epigenetic modifications across the genome) has been implicated in disease states, and targeting...DNA sequences. Non-editing CRISPR applications direct catalytically dead dCas9 fused to a variety of epigenetic... the catalytic domain of p300 acetyltransferase increases levels of H3K27ac histone modification at specified...Preps for Epigenetic Modification Why use epigenetic modifiers? Epigenetic modification is certainly not ...

... systems to engineer better and faster ABEs (Fig. 2). These systems allow for rapid continuous protein...and less stringent phage selection. Figure 2: Phage-assisted evolution of base-editing activity... specific DNA base changes and consist of a catalytically impaired Cas protein (dCas or Cas nickase) fused... SpCas9. In order to expand the gene-editing applications and targeting scope of ABEs David Liu’s lab ...through many rounds of mutations, selection, and replications per day.  The PACE and PANCE systems use M13...

...certain states of interest. For example, in Figure 2 below we present a two-input, five-state GRSM register...linking RSM inputs to endogenous gene expression (2). Our paper (1) explains how to arrange attB-attP...Science. 353, aad8559 (2016). PubMed PMID: 27463678. 2. Green, Alexander A., et al. "Toehold switches: de-novo-designed...and attP recognition sites on the register and catalyzes their recombination, thereby changing the state...performed on one register at a time. If a particular application makes it difficult to isolate single registers...state read-out method may not be suitable for applications that require minimal cellular perturbation. ...The input plasmid (high copy, ColE1 origin of replication), contains large serine recombinase genes downstream...

...metabolic engineering applications in Saccharomyces cerevisiae.” FEMS Yeast Res. 12(2) (2012): 197-214. PubMed...immediate early gene expression. Genes Dev. 1988 Jun;2(6):718-29. doi: 10.1101/gad.2.6.718. PMID: 2843425...Targeted Gene Expression in Drosophila.” Genetics 186(2) (2010): 735-55. PubMed PMID: 20697123. PubMed Central... also been developed which don’t fit in either category, but those are a subject for a future post! ... GAL80 under different promoters and create sophisticated patterns of UAS-driven gene expression. To further... PMCID: PMC2942869. Find plasmids from this publication at Addgene. del Valle Rodriguez, Alberto, Dominic... PMCID: PMC2883883. Find plasmids from this publication at Addgene. Wei, Xing, Christopher J. Potter...

...without leaving behind any accessory DNA sequences [2], [3]. Simply put, the piggyBac™ transposon containing...with a Footprint-Free™ genome edit.   Figure 2: Editing DNA using the piggyBac™ transposon selection...PMID: 26211958. PubMed Central PMCID: PMC4663986. 2.  A. M. Singh, V. V Adjan Steffey, T. Yeshi, and D...piggyBac™ technology can be used for numerous applications, including transgenesis, gene-trap screens, ...of the piggyBac™  transposase/transposon gene modification system and the scarless, Footprint-Free™ removal...technology that may be used for a variety of research applications, including making transgenic cell lines and ...PMCID: PMC2567143. 6. S. Radecke, F. Radecke, T. Cathomen, and K. Schwarz, “Zinc-finger nuclease-induced...

...lab collection for more) pLexA and pACT2.2 - Yeast 2-hybrid plasmids Fly UAS, MT Targeted... expression, transgenics and genome modification Species-Specific Expression If you want to drive...His or optimized YFP tags for purification or localization experiments Yeast...for bacterial expression (Gateway) GST Protein Purification pEBG - N-terminal GST for mammalian... packaging and expression. See our dedicated lentiviral plasmid page . Retroviral Easy and ...shRNA expression, transgenics and genome modification Element Details Representative Empty ...

... pCRISPomyces-2 contains Cas9 and a gRNA cassette. The simpler system of pCRISPomyces-2 displays a higher...and a subsequent curing protocol that requires only 2-3 hours incubation. E. coli and T. citrea scarless...four major components including (1) a CRISPR RNA, (2) four proteins forming the QCascade DNA-targeting ...for successful modifications, efficiency can be low, especially for larger modifications. What’s the solution...several potential CRISPR applications. First, CRISPR can be used to target locations in the genomes of pathogenic...tools available for bacteria and some of the applications for which they’ve been used. The beginnings ... of Enterobacteriaceae, without the need for modification. This finding suggests that the system may be...

... tropism and axonal transport of AAV serotypes 1, 2, 5, 6, 8 and 9 in the mouse brain. PLoS One. 2013 ...PMID: 24086725. PubMed Central PMCID: PMC3785459. 2. Krashes MJ, Koda S, Ye C, Rogan SC, Adams AC, Cusher... preps. Cas9 is the headliner of most CRISPR applications and can be expressed from your cell line of ...Find Ready-to-Use Lentivirus Find Ready-to-Use AAV Catch up on Chemogenetics ...

...nonsense-mediated decay. Although Nelson et al. observed only 2% genome editing in one experiment, they found the ... cells throughout the body, especially stem cells 2. Remove any immunogenicity of the AAV vector Once ...Science. 2015 Dec 31. doi:10.1126/science.aae0169 2. Long C, Amoasii L, Mireault AA, McAnally JR, Li H...setting. CRISPR is often used for animal germline modification, to repair or add in disease-causing mutations...that AAV-CRISPR does not cause obvious germline modification, an important finding given the controversy ...Blog Learn How CRISPR Could Be Used to Treat Cataracts and Cystic Fibrosis Read How Homology Directed...

...The groups in these programs typically consist of 1-2 mentors and 3-5 mentees who meet together for an 8... together. If you all work at the same place, a 1-2 hour lunch on a regular schedule might work. If not...This is a chance to practice leadership and communication skills. The leader also provides materials such...

...residues, which can be found throughout the antibody. 2) Endogenous cysteines, such as those that make up ...Detecting antigens by quantitative immuno-PCR. Nat Protoc 2:1918–1930. https://doi.org/10.1038/nprot.2007.267 ...These properties allow for efficient signal amplification, making biotin conjugated antibodies good options...especially if you start to consider clinical applications). But there are a few other classes that we ...proximity ligation, immuno-PCR, and single-cell applications (Hegazy, et al., 2020; Niemeyer, et al., 2007...also conjugate antibodies directly to beads for purification purposes.  Coupling up - antibody conjugation...added to antibodies post-translationally. 4) Modifications made to antibodies, such as inclusion of non-canonical...

...that increase cancelled. Rounding out the remainder, 2% of postdocs who weren’t going to get a salary increase...with their plans, and 17 have cancelled their plans. 2 of those raising - the University of Illinois and ...days prior to the implementation date, our data indicated that, while 69% of all postdocs were expected ...sent, with the lack of transparency and poor communication of their plans (many postdocs - and in some ...to reach out to institutions with a statement indicating that we encourage them to keep raising salaries... little correlation with cost of living at the location of the institution. Continuing efforts to gather...Director of Future of Research. He is interested in advocating for and with junior scientists, to help them ...

... PMC4096992. Find plasmids from this publication at Addgene. 2. Tang, Jonathan C.Y., et al. “Cell type-specific...successfully regulated expression of channelrhodopsin-2, commonly used in optogenetics, opening up the possibility...repurpose popular GFP reporter lines for more sophisticated experimental manipulations, saving the time ..., 98% of TdTomato expressing cells were GFP+, indicating a robust, but specific system. T-DDOGs also successfully...systems are easily adaptable to neuroscience applications, including optogenetics, and they should make...Could Cas9-DOG be developed soon to allow genome modification only in a small subset of cells? Other proteins...PubMed PMID: 26258682. Find plasmids from this publication at Addgene. 3. Kirchhofer, A., et al. “Modulation...

...plasmid kit consists of 20 plasmids encoding 14 alpha, 2 beta, and 4 gamma subunits of the heterotrimeric G...activation via bioluminescence resonance energy transfer 2 (BRET2). Check out our blog post on GPCR signaling...constructed plasmids that can be used in peroxidase-catalyzed proximity labeling to quantifiably measure GPCR...In this technique, the receptor fused to APEX2 catalyzes the biotinylation of proteins near the GPCR. These...Multidimensional Tracking of GPCR Signaling via Peroxidase-Catalyzed Proximity Labeling. Cell 169:338-349.e11 . https...Kadowaki S, Haga T, Takaesu H, Mitaku S (2002) Identification of G protein-coupled receptor genes from the...

...linear fashion. This implies that reducing the power 2-fold does not reduce photobleaching by half. How exactly...prolonged time). Other illumination strategies, such as 2-photon-excitation, selective plane illumination, TIRF... localized fusion proteins and do not require dedicated plasmids or constructs. It should be noted that...reached at high power may not translate to real applications in which far less power is used. For example...insight into the photostability in the intended application (Goedhart et al, 2012). The third issue relates...Selecting a photostable protein The issues with quantification of photobleaching rates can be summarized as...

...11 and Cas13 are 1). non-specific RNA cleavage and 2). cell toxicity. Cas7-11 does not display collateral...Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell, 163(3): 759-771 (2015). 10.1016...a defined site to form a clean DNA break, Cas3 locates its target sequence and initiates single stranded... out large regions of DNA and has anti-viral application potential as a result. Fast facts – PAM requirement... has been proposed to be useful for the same applications (RNA knockdown, RNA editing, etc.). The main...

...more tips on using social media as a scientist here. 2) Public engagement - Participate in science festivals...science communication are as varied as the field itself. Among other things, science communication comprises...certain aspects of art and education. If you suspect that science communication might be for you, don’t ... science education Do you aim to reach young people? Try networking with your local education community... Training – Get a formal education Some people get into science communication by taking a media or journalism...project. Science communication opens doors Getting involved in science communication has been the most...career researcher looking to break into science communication? Everybody goes about this in their own way....

...this quick trick. Just flip, and press! Lab tip #2: Tube flick trick When combining reagents in the lab...With barcode tracking, you will always be able to locate materials quickly and easily. Learn more about ...Additional Resources on the Addgene Blog New Educational Resource: The Addgene Videos Page 5 Science Rap...