...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...

...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...

...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...

...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...same time as, or slightly 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...

...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...

...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...

...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 ...

...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...

...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 ...

...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....

... 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...

... 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...

...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...

...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...

...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 ...

... 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...

...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 ...

...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...

...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...

...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 ...