Skip to main content
Addgene

Enter a sequence to perform a BLAST-based search of Addgene plasmid sequence databases.

In Category

We narrowed to 612 results for: SCR

Showing: 51 - 100 of 612 results

  1. CRISPR Methods for Bacteria: Genome Engineering, CRISPRa, CRISPRi, Base Editing, and More

    Type
    Blog Post
    Published
    Sept. 28, 2020, noon
    ...proteins involved in the activation of transcription including transcription factors, phage proteins, and RNAP...citrea scarless editing plasmids Sheng Yang’s lab describes a two-plasmid system that combines recombineering...array, CRISPR-transposons can be multiplexed. Transcriptional repression (CRISPRi) in bacteria As RNA interference...targeted to a promoter or gene body can repress transcription by physically blocking the elongation complex...complex from binding the DNA or extending the transcript. Different Cas orthologues and subtypes function ...regulatable CRISPR system effectively silenced transcription of one or more genes in E. coli. and in a mammalian...reliably knockdown expression of multiple target transcripts simultaneously. Marraffini lab's CRISPRi/a plasmids...

  2. Prime Editing: Adding Precision and Flexibility to CRISPR Editing

    Type
    Blog Post
    Published
    Jan. 13, 2025, 9:49 p.m.
    ...tools. Scroll down to see a table summarizing all of them! PE2: Improving reverse transcriptase efficiency...consists of a Cas9 nickase fused to a reverse transcriptase. The Cas9 nickase is guided to the DNA target...hybridizes to the target site, and the reverse transcriptase domain copies the desired edit, directly polymerizing...Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase to the C-terminus of Cas9 H840A nickase (PE1...efficiency Building upon prior reverse transcriptase research, the Liu lab created and evaluated 19 PE1 variants...inventors kept optimizing it. Using a pooled CRISPRi screen, the Liu lab discovered that cellular mismatch ...PE2-PE5 strategies. PEmax contains a reverse transcriptase with a sequence optimized for human codons; ...

  3. 9 tips for a successful postdoctoral experience

    Type
    Blog Post
    Published
    Oct. 9, 2018, 12:21 p.m.
    ...and the amount of time spent shopping a manuscript. Manuscript writing can take weeks to a couple of months...for and how can you do it well? Below, I briefly describe some tips for a successful postdoc fellowship..... Now work backwards. To publish a submitted manuscript in a high-profile journal typically takes 6-12...for a Group Leader position, key first author manuscripts need to be ready to write by Year 3.5. Thus, ...end goal of having sufficient data to write a manuscript. Consider questions like: What figures are missing...model?  PFs need to be regularly thinking about manuscripts. The other items will follow. If you get more...validated? How will the results fit into a future manuscript? You could easily spend a day or a week planning...

  4. Hot Plasmids Spring 2024

    Type
    Blog Post
    Published
    May 23, 2024, 1:15 p.m.
    ...CRISPR screens By Emily Bentley Building on their CROPseq method for optical pooled CRISPR screening, Paul...Multiplexed perturbation and decoding in pooled CRISPR screens Improved prime editor PE7 Antibodies to facilitate...multiplexing approach to Cas9-based pooled CRISPR screens called CROPseq-multi (Walton et al. 2024). This...enrichment, single-cell sequencing, and optical pooled screens in human cells. Addgene offers vectors for CROPseq-multi... with tRNAs, which are cleaved out of the RNA transcript by endogenous processing enzymes (Figure 1). ...processing only occurs on the RNA polymerase III transcript, the sgRNAs were encoded on the lentiviral minus...multiplexed perturbation and decoding in pooled CRISPR screens. bioRxiv 2024.03.17.585235; doi: https://doi.org...

  5. How to Design Your gRNA for CRISPR Genome Editing

    Type
    Blog Post
    Published
    Sept. 24, 2020, 1:15 p.m.
    ...engineer specific DNA edits and to perform functional screens to identify genes involved in a phenotype of interest...the need for single cell cloning and subsequent screening for successful edits. This is a very time consuming... modulating gene expression at the level of transcription – CRISPRa (activation) and CRISPRi (inhibition...most-efficacious to target a ~100nt window upstream of the transcription start site (TSS), while for CRISPRi, a ~100nt...of producing the guide (synthetic, in vitro transcription, or lentiviral delivery) can affect the accuracy...examined whenever possible when using knockout or transcriptional modulation approaches. Avoiding off-target ...Further, large-scale datasets of hundreds of genetic screens using genome-wide libraries have shown high concordance...

  6. Tips for CRISPR Gene Editing in Mice

    Type
    Blog Post
    Published
    June 28, 2016, 10:59 a.m.
    ...during your in vivo work is to screen you gRNA in vitro first. In vitro screening is a simple and fast method...has been quite successfully adapted for use in screening experiments as well). Therefore, if you’ve started...Several companies supply gRNA sequences, such as GenScript, ThermoFisher Scientific and OriGene, or you can...previously validated gRNAs at Addgene. In vitro screening for the best gRNA Figure 1: Validation ...gRNAs to Target Mouse Genes To begin the in vitro screening process, you should first develop primers to the...embryos to develop. Once they are born, it’s time to screen them for your desired edit. Good news! Remember...no mutation, costing money and space. As a rule, screen all of your pups unless you have an enormous litter...

  7. Clear and Accessible Writing

    Type
    Blog Post
    Published
    Feb. 15, 2024, 2:15 p.m.
    ...the use of alt text to describe images in online spaces. Alt text is used by screen readers to provide visually...Students Writing Scientific Manuscripts Tips for Improving Your Next Manuscript...example. Remember, our goal is to use words to describe the image in a way that helps visually impaired... your alt text, use all of this information to describe the image helpfully. You and your readers are ...learned a lot from listening to others' alt text descriptions, and we’d love to hear yours as well.  More ... You can use them to help you write an entire manuscript, or you could use them to write alt text for ...for any image you’re struggling to describe in the results. And many journals accept alt text, so you can...

  8. Teaching an Old DOG New Tricks: Controlling Protein Activity with GFP

    Type
    Blog Post
    Published
    Nov. 24, 2015, 3:30 p.m.
    ... 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...Using nanobodies to create GFP scaffolds for transcriptional activation The project began with a frustration...GFP-labeled cells. Once Tang and Cepko found a description of GFP-binding nanobodies, the project really...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...T-DDOG results showed that GFP could regulate transcription in a cell-type specific manner, and Tang and...

  9. Corralling and Cornering the Goldilocks Antibody

    Type
    Blog Post
    Published
    Aug. 9, 2022, 1:15 p.m.
    ...specific markers. The Screening Criteria While choosing which antibodies to screen, we kept in mind our...We therefore use both rAb and mAB in our screening. To screen the antibodies, we have developed a criterion...Initiative Cell Census Network, the Wu lab will be screening over 3,000 antibodies for use in mice and human...to see the reproducibility and accuracy while screening. We also compare different clones of the same ...multiple people in the lab conduct “independent screening”, where they look at the same section and determine...reproducibility, each antibody is independently screened twice, confirming it will produce the same results...available 2D/3D mouse atlas from antibodies that we screen. This atlas will enable scientists to have an intuitive...

  10. Truncated gRNAs for Regulating Gene Expression

    Type
    Blog Post
    Published
    Jan. 10, 2017, 3:37 p.m.
    ... then be fused to effector domains such as transcriptional activators or repressors. These fusion proteins...Consequently, fewer genetic tools for targeted transcriptional and epigenetic regulation have been validated...Furthermore, among those Cas9 orthologs that have been scrutinized, the majority have been shown to exhibit more...inexpensively generate a potent Cas9-based transcriptional regulator without making any functional changes...contains an RNA adapter capable of recruiting a transcriptional activator, robust regulation of gene expression...incorporated into the truncated gRNA recruits a transcriptional activator to the Cas9-gRNA complex. When this...complex is directed to a target upstream of the transcriptional start site (TSS) of a gene of interest, it ...

  11. An “elegans” Approach to Better CRISPR/Cas9 Editing Efficiency

    Type
    Blog Post
    Published
    Dec. 17, 2020, 1:30 p.m.
    ... ready for experimentation. In 2014, several manuscripts published in Genetics detailed approaches in ...variable amounts of experimental manipulation and screening.  A co-selection strategy for CRISPR editing in... with inefficient sgRNAs in my initial direct screening efforts, which were similar to the approach detailed...low efficiency (0.13%) and laborious handling (screening 768 F1 animals). Recovering rare editing events...conditional-lethal mutation to identify edits with minimal screening effort. Selecting for repair of a lethal mutation...to identify a single 2xFLAG knock-in by direct screening. The center images depict the power of lethal ...parental animal, are rescued progeny, which makes screening extremely rapid. This stringent selection allowed...

  12. 22 Hot Plasmid Technologies from 2014

    Type
    Blog Post
    Published
    Jan. 6, 2015, 5:21 p.m.
    ...large scale functional screens. For more information on these new CRISPR screening tools, see our CRISPR... Lentiviral CRISPR libraries for knockout screening New systems have been developed and deposited ...CRISPR-Cas technology to perform genome-wide knockout screens. These vectors and sgRNA libraries expand upon ...CRISPR Libraries Enable Genome-Scale, Knockout Screening. Also, check out our updated CRISPR-Cas resources...minimizing confounding secondary effects? An approach described as “knocksideways” (a British idiom for “taking...Chrimson Through de novo sequencing of 127 algal transcriptomes, as well as further optimization through engineering...two-color optical excitation of neurons. The paper describes five previously unknown channelrhodopsins from...

  13. Plasmids 101: Gene, enhancer, and promoter trapping

    Type
    Blog Post
    Published
    May 2, 2023, 1 p.m.
    ...isolated and screened. Depending on the marker, traps can be used as high-throughput screens to introduce...Each trap uses a slightly different approach (described in detail below), but fundamentally the reporter...models. Why traps are useful Traditional genetic screens employ various on/off switches to measure the function...circumstances, trap methods may be the preferred screening option. Promoter traps Promoter trap systems ...integrate within an exon of a gene to generate a transcriptional fusion. This may disrupt the endogenous function...spliced into the gene product resulting in a transcriptional fusion, much like promoter traps. While this...likely disrupt the gene it monitors due to transcriptional fusion.   Examples of promoter, enhancer...

  14. 10 Great Guest Posts We're Thankful For

    Type
    Blog Post
    Published
    Nov. 18, 2016, 3 p.m.
    ...Detect Zika Virus The RNA-based toehold switches described in this post are engineered to bind RNA sequences...Boston University School of Medicine, this post describes a tool you can use to study basic virus biology...sequences required for viral replication and transcription as well as reporter genes. To learn how you ...researcher in the Silver Lab at Harvard University, describes R-bodies, “force generating protein machines that... Connie Cepko’s Lab at Harvard Medical School describes how AAVs function and indicates important things...CombiGEM and CRISPR for Combinatorial Genetic Screening CombiGEM is a technology that can be used to quickly...barcode small libraries of CRISPR gRNAs to use for screening experiments. Get the full details on how to make...

  15. Single-cell tracking of lineage and identity with CellTag

    Type
    Blog Post
    Published
    May 9, 2019, 1:15 p.m.
    ...Direct reprogramming describes the process where differentiated cells are turned into a cell type of ...cell technologies, such as single cell RNA-seq (scRNA-seq), now allows scientists to identify the gene...cells that successfully reprogram. Unfortunately scRNA-seq only captures the gene expression of cells at...identifiers act like a timestamp and help demultiplex the scRNA-seq data and build multi-generational cell lineage...reprogrammed into iEPs by retroviral expression of transcription factors Foxa1 and Hnf4α, a process that like...reprogramming (CellTagMEF), at the end of the three-day transcription factor reprogramming treatment (CellTagD3), ...three days, a sample of cells was collected for scRNA-seq analysis and the remaining cells were maintained...

  16. How to Keep a Lab Notebook for Bioinformatic Analyses

    Type
    Blog Post
    Published
    June 2, 2016, 2:30 p.m.
    ...commonly used scripts: If you edit a general purpose script for a single use, save the script as originalName_descriptionOfEdit...be in the scripts that you run. Rather, this is where you outline your logic and the scripts and input...immediately write a text document with a brief description of the directory contents, at a minimum. This...for all of your analyses. The same is true for scripts. Naming conventions Give every experiment a number... remains in tact! Take notes Comment in your scripts: Comments on what a given line of code does are...

  17. A Look at Addgene's QC Process

    Type
    Blog Post
    Published
    May 6, 2025, 1:15 p.m.
    ...effort into our quality control (QC) processes to screen for and reduce these errors. These processes allow...types of data for each of their plasmids, from descriptions of key components of the plasmids to partial...process ensures that any potential sequence discrepancies can be caught prior to distribution. Catching...issue? At Addgene, a QC issue is defined as a discrepancy that we believe may affect the function of the...decisions when analyzing the wide variety of discrepancies encountered in plasmids deposited to Addgene...resolve. Sequencing and assembly issues Sequence discrepancies are not always a result of mutations in the ...will contact the depositing lab to review the discrepancies. Often, the identified issues will not affect...

  18. Drew Endy Introduces the Biobrick Public Agreement Plasmid Collection

    Type
    Blog Post
    Published
    Dec. 12, 2013, 3:35 p.m.
    ...with a publication in Science describing the development of “transcriptors,” transistor-like digital genetic...cellular computing. These logic gates built from transcriptors are available at Addgene as a BIOFAB kit comprised...comprised of well-characterized bacterial transcription and translation initiation elements. Open science...with the central dogma - replication of DNA, transcription of DNA to make RNA, translation of RNA to make... and observed that we humans find it useful to describe and interact with the world using words like OR...The future of synthetic biology Addgene: You've described the pieces in these kits as the beginnings of ...systems today who are building 10-gene systems from scratch. (They may be building genomes, but that's just...

  19. Uncovering Ancestral Bias in CRISPR Research

    Type
    Blog Post
    Published
    Nov. 29, 2022, 2:15 p.m.
    ...genome-wide screens, introducing bias into all work based on these and any other CRISPR screens using the..., race. Many researchers do large-scale CRISPR screens in cancer cell lines, looking to uncover genetic...targets and related biomarkers . Data from such screens are often uploaded to the Cancer Dependency Map...Boehm labs pulled data from more than 1000 CRISPR screens in DepMap to see if they could identify the ancestry... American, Africa, or South Asian) used in the screens based on the cells’ germline genetic variations...about 90% of the cell lines used in DepMap CRISPR screens are of either European or East Asian ancestry, ...actually reference?  The CRISPR guides used in the screens they analyzed were designed to have a perfect match...

  20. Isolating Genomic Regions of Interest with the CRISPR System

    Type
    Blog Post
    Published
    May 30, 2019, 12:44 p.m.
    ...dCas9 has focused on transcriptional activation using a fusion to a transcriptional activator such as VP64...repression of transcription through binding a promoter region to inhibit association of transcriptional activators...region In 2013, the Hodaka Fujii Laboratory first described a method to purify a specific genomic region using...locus of interest, the promoter region of the transcription factor IRF-1. Immunoprecipitation, using an ...plasmid. Experimental use Since the initial report describing CRISPR-mediated purification of a specific genomic...Tapscott Lab identified regulators of DUX4, a transcriptional regulator that when inappropriately expressed...CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes." Cell 154.2 (2013): 442-451. ...

  21. Plasmids 101: Colony PCR

    Type
    Blog Post
    Published
    May 12, 2016, 2:30 p.m.
    ...Molecular cloning requires some method of screening colonies for the presence of an insert. Traditionally...backbone. This type of primer pair is also great for screening clones created with the same backbone but that...is, allowing you to use the same primer pair to screen for the presence of many different inserts. The...test your colony PCR primers before using them to screen colonies. The best way to do this is by using your...insert and if it's in the plasmid Can be used to screen clones that were created with the same backbone... background, the more colonies you will need to screen.   Saving clones for later culture: At this point...expected results for the three primers previously described. When using insert-specific primers (1), positive...

  22. Modulate the Activity of 17 Signaling Pathways with One Kit!

    Type
    Blog Post
    Published
    March 22, 2016, 2:30 p.m.
    ...a plasmid kit designed to do exactly that. As described in the journal Science Signaling in December 2014... to each to make the library usable for pooled screens. Applications of the cancer pathways ORFs kit Wood...and his colleagues developed the kit for use in screening for pathways that drive resistance to drugs in...the only way to use the kit. “You could use it to screen for pathways that confer any phenotype of interest...drug resistance,” he said. “It could [be used to screen for] morphological features, migration, or survival...use our libraries to do things - sometimes drug screens and sometimes totally different experiments we ...Tracing Read How CRISPR Can Be Used in Genome-Wide Screening Browse 28 Hot Plasmid Technologies from 2015 ...

  23. Designing Your Chalk Talk for the Academic Job Interview

    Type
    Blog Post
    Published
    March 12, 2019, 1:37 p.m.
    ...assays for your screen result may or may not actually help. Ultimately, if you cannot describe the likely ...of experiments or a laundry list of molecules to screen has unclear utility. You cannot predict what will...cannot either. Therefore, you need to be able to describe experimental approaches in terms of the question...approach, anticipated outcomes, and preferably a description of the payoff if the aim is successful. Note ...the aims and clarify what type of aim you are describing. It's easy to be overly ambitious – a common ...distinguish mechanistic questions from open ended screens and data collection. Organize your research vision...accomplished in 5-10 years? You need to be able to describe what success looks like. If you are successful...

  24. CRISPR 101: Which Cas9 Do I Choose for My CRISPR Experiment?

    Type
    Blog Post
    Published
    Oct. 13, 2022, 1:45 p.m.
    ...targeting dCas9 to transcriptional start sites was sufficient to repress transcription by blocking its initiation...desired edit adjoined to the sgRNA, a reverse transcriptase, and a nCas9. While this system has been shown...Prime editor engaging target DNA. The reverse transcriptase polymerizes DNA onto the target strand based...been exploited to localize factors such as transcriptional activators and repressors to target genes. ...repression requires targeting dCas9 along with transcriptional repressors to the promoter region of the gene...associated box (KRAB) is a naturally occurring transcriptional repressor domain which is frequently fused ...activators consists of dCas9 fused to a single transcriptional activation domain - VP64.  Later generations...

  25. 15 Hot Plasmids from 2017

    Type
    Blog Post
    Published
    Jan. 9, 2018, 3:02 p.m.
    ...hot plasmids segment on the Addgene Podcast. Subscribe to the Addgene blog to get all the latest hot ...pluripotent stem cells) through the expression of transcription factors OCT3/4, SOX2, KLF4, and c-MYC is the... method for generating iPSCs to express the transcription factors from a polycistronic cassette. However...polycistronic cassettes and discovered reprogramming discrepancies, which they traced to a 9 amino acid N-terminal... did not lead to the observed reprogramming discrepancies. The polycistronic piggyBac transposon vectors...the RFPs developed from natural ancestors), and screened through libraries of new RFP variants for increased...beautifully demonstrate light-dependent Gal4 transcriptional activation and Cre recombination. Find PhoCl...

  26. Plasmids 101: Common Lab E. coli Strains

    Type
    Blog Post
    Published
    Nov. 7, 2014, 2:56 p.m.
    ...Dr. Theodor Escherich, the scientist who first described them in 1885. E. coli are mainly found in the ...mutations found in E.coli strains Gene(s) Description Functional Consequence dam DNA adenine methylase...methylate DNA lac Lac operon mutations Blue/white screening of clones   lacIq lac repressor overproduced...derivative. General cloning and storage, blue/white screening, leucine auxotroph. F- endA1 recA1 galE15 galK16...cloning and storage of common plasmids, blue/white screening. F- endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR...General cloning and plasmid maintenance, blue/white screening, partly restriction-deficient; good strain for...derivative. General cloning and storage, blue/white screening. F- mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74...

  27. Viral Genome Sequencing - A One Stop Shop for AAV Identity and Serotype Confirmation

    Type
    Blog Post
    Published
    April 23, 2020, 1:15 p.m.
    ...our AAV preparations. We recently published a description of the VGS process and use cases in Human Gene...ligation and tagmentation preparation approaches described previously require a preliminary double stranding...addition, we wrote an open-source custom Python script that uses the VGS data to determine the percent...recombination present in Cre-dependent vectors and a script that can confirm the serotype of AAV preparations...use this to our advantage by designing a Python script that looks for pre-defined serotype specific signature...signature sequences in the data. Unlike previously described thermodynamics-based methods of serotype confirmation...parallel and provide serotype data in minutes and the scripts  can be easily customized to detect other sequences...

  28. Summer SciComm: Preprints

    Type
    Blog Post
    Published
    Aug. 16, 2022, 1:15 p.m.
    ...is the rising use of preprints, or scientific manuscripts shared by the authors on a public server independent... their work publicly and immediately once the manuscript is drafted. Preprints were one of the key routes...at the same time as, or before, submitting the manuscript to a journal for formal peer review. Some organizations...unpublished results at a conference? Post your working manuscript as a preprint too and you can share all the details... use your work but don’t think they’ll have subscriptions to the specialized journals in your field? Post...only way to rigorously determine or ensure a manuscript’s quality. But even journal-based peer review ...giving authors the opportunity to strengthen the manuscript prior to submission. Preprints at Addgene Preprints...

  29. The Addgene Antibody Data Hub

    Type
    Blog Post
    Published
    Sept. 12, 2024, 1:15 p.m.
    ...contains a detailed description of the experimental conditions.     Figure 4: A screenshot of the materials...antibody worked under the conditions described above, and a short description of how the antibody performed....the submitted application, along with a brief description of the experiment. If that data was included ...conditions in which the data was generated, you can scroll down the page to the materials and methods section...The submission process is easy, with simple, descriptive fields, drop-down boxes when appropriate, and...

  30. Viral Vectors 101: The Retroviral Lifecycle

    Type
    Blog Post
    Published
    July 27, 2023, 1:15 p.m.
    ...reverse-transcribed DNA into the host cell’s genome and then use cell machinery for transcription and translation...structural proteins expressed from a reverse-transcribed piece of DNA called a “provirus.” This DNA is...(or host cell) by the viral protein reverse transcriptase (RT). In the case of a natural virus, both the...the protein and the genetic code for reverse transcriptase are packaged (Wu et al., 1997) inside of the...viral vectors, only the protein for reverse transcriptase (RT) is present in the capsid, as the engineered...is a turn of phrase — “pseudotransduction” — to describe the direct protein transfer of RT, which would...during uncoating. After uncoating, reverse transcription follows the release of the two strands of viral...

  31. Your Top Requested Plasmid in 2015!

    Type
    Blog Post
    Published
    Dec. 30, 2015, 3:30 p.m.
    ...about pooled library screening, read our recent blog post on Genome-wide Screening Using CRISPR/Cas9. If... population of cells as part of pooled library screens (1,2). The human and mouse GeCKO libraries, targeting...lentiCRISPR v2 is key to its efficient use in screening experiments; large amounts of virus are required...you get full library coverage in pooled library screens. Availability and Use LentiCRISPR v2 is available...Ophir, et al. "Genome-scale CRISPR-Cas9 knockout screening in human cells." Science 343.6166 (2014): 84-87... vectors and genome-wide libraries for CRISPR screening." Nature methods 11.8 (2014): 783-784. PubMed ...

  32. An Inside Look into Addgene's Dev Team

    Type
    Blog Post
    Published
    Jan. 23, 2020, 2:45 p.m.
    ...methodology of software development called Scrum. The Scrum framework is based on an iterative and collaborative...team because we are using the Scrum framework or we are using the Scrum framework because we are highly...collaborative approach to development. Scrum teams plan and break up work into small, well defined chunks...needs of the teams at Addgene. Another advantage of Scrum is that it provides transparency to everyone involved... on a new feature. We start with a story that describes the feature and what the criteria is for accepting...that the clarifications lead to the story being scrapped or broken up into smaller stories. Since this ...

  33. Let There Be LITE Plasmids

    Type
    Blog Post
    Published
    Nov. 21, 2013, 3:56 p.m.
    ...of customizable DNA-binding domains based on transcription-activator-like effectors (TALEs) as anchors ... thaliana. The results are light-inducible transcriptional effectors (LITEs) designed to bind specific.... Genes under LITE control showed a rise in transcription in as little as 30 minutes after light stimulation...News & Views Andreas Möglich and Peter Hegemann described several advantages of the new system: "First,... universally abundant. Second, induction of transcription occurs within minutes of light exposure. Third...really flexible and modular,” Konermann said. “Transcriptional activators are easy to expand or combine with...al. "Optical control of mammalian endogenous transcription and epigenetic states." Nature. 500, 472–476...

  34. The Fluorescent Vegetables in Aptamer Soup

    Type
    Blog Post
    Published
    Jan. 29, 2019, 2:27 p.m.
    ...quantify RNA transcription by RNA Polymerase III. By expressing Corn under promoters for transcripts uniquely...tagging RNA sequences with an aptamer and RNA transcription by placing the aptamer downstream of a known... Addgene’s Plasmids 101: Aptamer Fluorophores describes the enrichment process used to evaluate oligonucleotides... of colors, so do aptamers! Corn was recently described in 2017 (Song et al., 2017). Corn naturally dimerizes...uniquely transcribed by RNA Polymerase III (5S, tRNA and U6) and measuring Corn fluorescence by flow cytometry... Red and orange fluorescent aptamers Corn was described in the same study as Red Broccoli and Orange Broccoli...Wenjiao, et al. "Imaging RNA polymerase III transcription using a photostable RNA–fluorophore complex....

  35. Plasmids 101: Gibson Assembly and Other Long-Homology Based Cloning Methods

    Type
    Blog Post
    Published
    March 1, 2016, 3:30 p.m.
    ... friends on the Cambridge 2010 iGEM team that describes the basics of Gibson assembly as a parody of “...assembly The Gibson assembly technique was first described by Dr. Daniel Gibson and colleagues at the J. ...typically fairly high, so there is usually no need to screen a large amount of colonies. Excluding the time ...combined with the popular CRISPR technology and is described in a recent publication by the Lockey lab (Wang...followed by the standard Gibson assembly techniques described above, this resulted in the direct and seamless...using Gibson Assembly along with CRISPR was also described recently (Jiang et al. 2015), in which very large...challenging. Other homology based technologies We've described Sequence and Ligation Independent Cloning (SLIC...

  36. Hot Plasmids - June 2020 - Barcoded CRISPR Library, Sparse Cell Labeling, Calcineurin Reporter, and DNA Staining Dye Alternative

    Type
    Blog Post
    Published
    June 2, 2020, 1:15 p.m.
    ... Figure 1: Initial steps in the CRISPR-pooled screening using sgRNAiBAR. Image from Zhu et al., 2019.  ... Peking University has developed a new CRISPR screening method that makes use of guide RNAs with internal...false-positive rate discovery rate, conventional CRISPR screens require a low multiplicity of infection (MOI) during... annotated human gene, it enables genome-wide screening with a high MOI, which is beneficial for a low... calcineurin dynamics and can serve as a drug-screening platform to identify new antifungals that target... assay, and are not suited to high throughput screening, calcineurin dynamics measurements, or monitoring...Sabrina Büttner’s lab designed a calcineurin transcriptional reporter consisting of a quickly-degraded destabilized...

  37. A New Optogenetic Tool Based on AraC Controls Gene Expression with Blue Light

    Type
    Blog Post
    Published
    June 17, 2021, 1:17 p.m.
    ...from one that represses transcription to one that activates transcription.However, this system lacks spatio-temporal...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...with AraC,” says Di Ventura. BLADE activates transcription with the flick of a light switch and shuts off...

  38. Recombinase-based State Machines Enable Order-dependent Logic in vivo

    Type
    Blog Post
    Published
    July 28, 2016, 2:30 p.m.
    ...the transcribed gene and its 5’ UTR. Various self-cleaving hammerhead ribozyme parts are described in ...of an RSM is defined by the sequence within a prescribed region of DNA termed the “register”. Each chemical...registers within a sample using a metric that we describe in the Materials and Methods section of our paper...sensors do not necessarily have to be based on transcriptional regulation, as long as they can control recombinase...recombinase activity. For example, post-transcriptional recombinase activation via RNA toehold riboregulation...to read states by qPCR or Sanger sequencing as described in the Material and methods section of our paper... Multiple large serine recombinases have been described in the literature (5, 6) and may be substituted...

  39. Viral Vectors 101: Gamma-Retroviral Packaging Systems

    Type
    Blog Post
    Published
    Aug. 6, 2024, 1:15 p.m.
    ...lentiviral packaging methods. The three components described above (envelope, packaging, and transfer) are ...nomenclature of the plasmid backbone is meant to be descriptive of the components found within the backbone. ... Pol Precursor protein containing Reverse Transcriptase and Integrase components. Transfer cPPT... WPRE Woodchuck hepatitis virus post‐transcriptional regulatory element; sequence that stimulates...necessary for activation of viral genomic RNA transcription. R; Repeat region found within both the 5' and... 5’ LTR Acts as an RNA pol II promoter. The transcript begins, by definition, at the beginning of R,...rest of the provirus. 3’ LTR Terminates transcription started by 5' LTR by the addition of a poly ...

  40. Why and how to start studying chromatin (with Addgene's help)

    Type
    Blog Post
    Published
    June 12, 2018, 11:40 a.m.
    ...972.e23 Screen identifies bromodomain protein ZMYND8 in chromatin recognition of transcription-associated...times have you looked at a diagram depicting transcription, or DNA repair, or replication, or any number...researching enhancer elements that control the transcription of a gene a megabase away, or looking at the...plasmids containing the indicated gene, and you can subscribe to these pages to learn when Addgene receives ... articles reporting chromatin-related research describe the production of plasmids that might be useful...colleagues and guest bloggers have written posts describing new methods and tools for studying chromatin ...

  41. 15 Years of Addgene: The Top 15 Plasmids

    Type
    Blog Post
    Published
    Jan. 8, 2019, 1:54 p.m.
    ... lentiviral vector that contains a scrambled shRNA. This scrambled shRNA is used as a negative control...sequence (pSpCas9(BB)). The 2A-GFP allows for screening of transfected cells. Find pSpCas9(BB)-2A-GFP ...virus, making it ideal for CRISPR pooled library screening experiments. Find lentiCRISPR v2.(If you’ve peeked...envelope expressing plasmid pMD2.G. Find pMDLg/pRPE. scramble shRNA - This plasmid from David Sabatini’s lab...control for mammalian RNAi experiments.  Find the scrambled shRNA vector. pLKO.1 - TRC cloning vector - This...the necessary elements for packaging, reverse transcription, and integration needed for the production of...

  42. Plasmids 101: The protein expression toolbox

    Type
    Blog Post
    Published
    Feb. 16, 2023, 7:05 p.m.
    ...fused to a transcriptional repressor to your gene of interest. It will inhibit transcription instead of...stranded RNA molecules that degrade target mRNA transcripts. When transiently introduced, they can deplete...that, too! CRISPR activators (CRISPRa) are transcriptional activators fused to a dead Cas9. The system...tetracycline or one of its analogs will induce these transcriptional changes, and expression is tunable by titration...expression goes back to baseline) and occurs at the transcriptional level.     Fig. 2 Tetracycline...post-translational level, unlike most of the other mechanisms described. Fig. 3 dTag mechanism of protein degradation...

  43. Pooled Library Amplifications

    Type
    Blog Post
    Published
    May 30, 2024, 1:30 p.m.
    ...Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening, a protocol paper by the Zhang lab...library has inserts of different sizes (such as a transcription factor overexpression library) or is prone to...Re-transformation of the SPLINTR Libraries (PDF, 401 KB) Screening Zhang Lab - Multiplexed Overexpression of Regulatory...et al. Nat Protoc. 2017 with modifications Screening pCMV-Rep78/68 Scanning Saturation Mutagenesis...Amplification and Sequencing Protocol (DOCX, 32 KB) Screening Rinehart Lab Iterative Synthetically Phosphorylated...More resources on the Addgene blog Genome-Wide Screening Using CRISPR Preparing Pooled Libraries NGS Quality...

  44. xCas9: Engineering a CRISPR Variant with PAM Flexibility

    Type
    Blog Post
    Published
    March 28, 2018, 6:52 p.m.
    ...xCas9 for transcriptional regulation and base editing Hu et al. also engineered transcriptional activators...activating transcription. Sites containing NG, GAA, or GAT PAMs averaged 56-91% transcriptional activation...subunit ω. This construct is designed to activate transcription from the accessory plasmid (AP) encoding phage...could recognize a library PAM, it would induce transcription of gene III, permitting phage propagation and... of xCas9 3.6 and 3.7 shows that we have only scratched the surface of modifications that can be made ...

  45. Don’t FRET: Bimolecular Fluorescence Complementation Makes Visualizing Protein-Protein Interactions Easy

    Type
    Blog Post
    Published
    Aug. 27, 2020, 1:15 p.m.
    ...open reading frame (ORF) library to screen PPIs among 450 transcription factors in live fruit flies. Although..., kinase signaling, integrin signaling, and transcription factor interactions (Kerppola, 2006). One of...interactions between basic leucine zipper and Rel transcription factors (Hu et al., 2002). This would be more...large number of constructs for high-throughput screening of PPIs. For example, Bischof et al. (2018) generated...organisms lags behind. BiFC can also be used to screen for small-molecule inhibitors of a particular PPI...quantification (Kerppola, 2006, 2013). These are described in more detail below: Fusion protein construction...

  46. Capturing a Moment with Marc Zimmer’s “The State of Science”

    Type
    Blog Post
    Published
    July 30, 2020, 1:15 p.m.
    ...recent resurgence of “Do-It-Yourself Science” described in Chapter 3, and of course the rapidly changing... science.”  The three chapters in this section describe the challenges of trusting science in a “fake ... of the book, called simply “Old Science,” he describes the now infamous tale of Rosalind Franklin and...changed significantly over the last decade. He describes the detection of gravitational waves--a story ...collaboration from scientists around the world. The paper describing the waves’ detection had over 1,000 authors. ...This last section of the book, Future Science, describes the author’s thoughts on science’s biggest challenges... doesn’t need to. The problems and challenges described in The State of Science have not changed in “COVID...

  47. CRISPR Between the Genes: How to Experiment with Enhancers and Epigenomics

    Type
    Blog Post
    Published
    Aug. 9, 2016, 2:30 p.m.
    ...CRISPR screen to disrupt these enhancers. The third and final step is to conduct our CRISPR screen. We start...stem cells, and conclude with a targeted CRISPR screen that can disrupt these enhancers. A real-world ...“GPS for proteins”) for NANOG, an essential transcription factor in embryonic stem cells (ESCs). Your ...that have NANOG binding sites 2. Design a CRISPR screen to target and disrupt the NANOG enhancers 3. CRISPR...you’re interested in designing your own CRISPR screens for enhancers, check out the Appendix. Conclusion...established with Bowtie2 using the parameters first described in Kearns et al.: bowtie2 -f -x HG19_GENOME --...

  48. The CRISPR Software Matchmaker: A New Tool for Choosing the Best CRISPR Software for Your Needs

    Type
    Blog Post
    Published
    Nov. 3, 2015, 3:30 p.m.
    ...after the piñata burst, value was subjectively ascribed relative to the basic needs of each individual... few prior publications to go on, a submitted manuscript could only have been viewed as a significant ...There are 8 major categories found in the table, describing everything from basic to advanced functionality...the user is expected to interact with the tool (a screen shot and overview of the table can be found above...Software design elements that fall into this category describe how the user is expected to interact with the ...require working knowledge of the command line or scripting. Input flexibility: Software tools require some...re engaged in heavy CRISPR design (I’m talking screens and/or very large genomes), then it is worthwhile...

  49. 3 Tips to Improve HDR Efficiency for CRISPR Editing in Human Cells

    Type
    Blog Post
    Published
    Sept. 5, 2017, 1:58 p.m.
    ...genome editing with CRISPR/Cas9 in human cells was described in 2013 (Cong et al, Mai et al) we were very excited...find the 0.2-0.5% correctly edited alleles, when screening reasonable amounts of single cell clones. Furthermore... together with the intended sequence change and screen a few hundred clones to find the right one Step... few hundred. From these two steps, you’ll be screening a total of a few hundred clones - Still better...longer feasible to incorporate mutations without screening thousands of clones to find a positive one. So...thinking further about the distance relationship described above, we realized that, in addition to increasing... and neurovascular diseases. He worked on the described project while he was a Postdoc in Marc Tessier-Lavigne...

  50. Hot Biosensors 2022: Year-End Roundup

    Type
    Blog Post
    Published
    Dec. 6, 2022, 2:15 p.m.
    ... They use a unique and powerful high-throughput screen to select variants based on performance metrics...: JEDI-2P was identified from a high-throughput screen, selected for increased voltage response, brightness...a new preprint, Jared Toettcher and colleagues describe pYtags, a modular system for monitoring specific...developed using a high-throughput multiparameter screen (Koveal et al. 2022). Lactate is a key metabolite...cellular concentrations of lactate. The microfluidic screening platform developed here is also a promising approach...Figure 6: A) Schematic of BeadScan platform for screening candidate biosensor variants for performance under...Koveal D, et al. A high-throughput multiparameter screen for accelerated development and optimization of...
Showing: 51 - 100 of 612 results