...Strong insect promoter from baculovirus Constitutive  Commonly used in expression systems for insect cells...to replicate plasmids and make sure the cells maintains it. Here, we'll cover the promoter, which is necessary...responsible for initiating the transcription of your insert into RNA. In practice, the term "promoter" describes...regulatory factors: enhancers, boundary elements, insulators, and silencers; however, some "leakiness" of ...Strong hybrid mammalian promoter Constitutive  Contains CMV enhancer, chicken beta actin promoter, and...with Tetracyline or its derivatives.  Typically contains a minimal promoter with low basal activity and... promoter. Ac5 General expression mRNA Strong insect promoter from Drosophila Actin 5c gene Constitutive...

...                           California Institute of Technology             Johns Hopkins University                           ...                           National Institute for Physiological Sciences Broad Institute                                       ...for tagged and untagged expression of SARS-CoV-2 proteins in bacteria, yeast, and mammalian cells. We teamed...from the Michael J. Fox Foundation are used in Parkinson’s research.  The extensive resource from the Structural... determine the three dimensional structures of proteins.  The RESOLUTE Consortium collection enables research... collection, a ‘One-Stop Shop’ for Fluorescent Proteins, and plasmids from several labs at Florida State...

...problems? Instead of directly fusing a fluorescent protein to your protein of interest, you instead fuse it...possibility of long-term imaging studies. If you instead overexpress your protein at higher levels, you ...is at a very high concentration. Overexpressed proteins also have the potential to form aggregates and...to the synthetic SunTag scaffold. This scaffold contains 10-24 copies of the short epitope GCN4. GCN4 recruits...SunTag, they attempted single-molecule imaging deep inside the cell, in the nucleus and cytoplasm. Again, ... of SunTag compared to traditional fluorescent proteins Improved brightness and signal-to-noise ratio... fluorescent proteins...

...alarming results are due, at least in part, to insufficient and non-standard record keeping despite scientist...to capture ideas, write down certain notes or brainstorm, but we have to admit that paper-based record...functionalities that are hard to imagine on paper, such as instant search through all saved data and automated writing...maintenance and support.” Besides that, academic institutions tend to think about the long term, so they need... once they establish the system within their institution. In the study, respondents were also asked about...providers used to supplement their paper notebooks. Instead, all of the important information is consolidated... In addition, if you are working for a large institution or company, site licenses might be available ...

...own machinery to replicate the viral genome and proteins. This is, for the intrepid researcher looking ...Virus structure While all viruses have a genome inside a protein shell known as a capsid, some also have...can either follow central dogma (gDNA to RNA to proteins) or require an intermediary DNA replication step...transcribed into mRNA, directly translated into proteins, or, in the case of retroviruses, reverse transcribe...transcribed into mRNA before being translated into proteins. RNA viruses replicate their genomes using a variety...egg sac. (Okay, some enveloped viruses bud out, instead of lysing, but they do generally still kill the...replicative ability. The remaining particle, which retains the protein coat (and envelope), has a “gutted”...

...please use one of these methods to ensure your insert retains the 3' A overhang: Use a mixture of proofreading...below, the “A” overhang on the blue PCR product insert comes from using Taq polymerase for the amplification...these kits also often have the topoisomerase site inserted into a beta-galactosidase cassette allowing a ...clones. Once you introduce your 3’-end “A” overhang insert, the magic of TOPO cloning happens. Basic procedure... Colonies: You can confirm the presence of your insert by PCR, restriction digest, or sequencing. Pro ...concentration or you are cloning an extremely large insert. Since the standard ligation reaction is fairly...PCR Use REPLACR Mutatgenesis to Easily Generate Insertion and Deletions in a Plasmids Learn How to Verify...

...Plasmids 101: E. coli Strains for Protein Production Addgenie Julian Taylor-Parker explains why you might want...Homology Directed Repair Addgenie Chari Cortez explains some of the basic science behind homology directed...production and gives a quick rundown of some of the strains you can use to express your protein of interest...find a successful clone? Addgenie Jessica Welch explains how Blue-White screening could be a potential ...Addgene Science Communications Intern Mary Gearing explains how Cas9 homolog, Cpf1, may make it easier to ... able to help you. Head Addgenie Joanne Kamens explains what career coaches do and offers up some career... to use with CRISPR as the cargo. Mary Gearing explains some of the clever ways to successfully combine...

...Oliver Griesbeck of the Max Planck Institute for Neurobiology has been working on genetically encoded...used for ratiometric in vivo imaging. Griesbeck explains, while these Twitch sensors are always bright,...particular advantage for observing events taking place inside cells that are on the move, such as T lymphocytes...Eindhoven has similar interest in FRET-based sensor proteins, particularly for the intracellular imaging of... should require the simple swapping of binding domains, in practice optimizing the sensors involves plenty...solution: they devised self-associating fluorescent domains whose association is disrupted in the presence ... of mOrange2 and mCherry as donor and acceptor domains. He says, these new colors might now enable observation...

...founder, Eric Perkins, is Addgene’s Director of Science Product Management and explains the origins of ABC: ... favorite books - both scientific and not - to inspire you to read a little more this October. Science..., examining relationships between microbes and insects, mammals, and plants, among others. I particularly...lost for an age) fight in the Napoleonic Wars and against something even more sinister. 1000 pages long. ...and with the events of the times."     - Aliyah Weinstein, Marketing and Communications Manager Addgene's... Aliyah Weinstein...

...blog Read our fluorescent proteins blog posts Download the fluorescent proteins 101 eBook Read our plasmids...cascade of caspases, protease enzymes that cleave proteins. Executioner caspases are activated last in the...Zhang et al., 2019). How does FlipGFP work? GFP contains 11 β-strands and one central ɑ-helix that can ...10 (β10), and (3) β-strand 11 (β11). Each part contains components and residues that are necessary for...specific protease that is commonly used to cleave proteins. This protease reporter based on sfCherry however...in FlipGFP can be applied to other fluorescent proteins to create an array of multicolor reporters that...fluorescent protein fusions Browse the fluorescent proteins plasmid kits ...

...pMinDisplay vector. GPI contains a C-terminal glycosylphostidylinositol anchor. SGZ contains a PDGFR transmembrane...update our iGluSnFR post! Here, we look at the origins of the system and explore advances that have happened...happened since our original 2018 blog post. The origins of iGluSnFR   iGluSnFR was developed in 2013 by...an increase in fluorescence intensity from the inserted GFP. After characterizing iGluSnFR in vitro, showing...localization through different C-terminal anchoring domains The original iGluSnFR in the pMinDisplay backbone...introduce iGluSnFR3 in two additional anchoring domains – GPI and SGZ. Fig: 4 Sequence schematics...Fluorescent Protein Biosensors Browse All Fluorescent Proteins Find AAV Tools References Marvin, Jonathan S....

...structural component of atherogenic lipoproteins, shuttles lipoproteins through the circulatory systems, ...LipoGlo: A reporter for measuring ApoB bound lipoproteins Article contributed by Leah Schwiesow A leading...cardiovascular disease is driven by atherogenic lipoproteins that accumulate within the vascular wall. ApoB...formation of atherosclerotic plaques. ApoB bound lipoproteins (ApoB-LPs) are indirectly measured through their... Steven Farber and colleagues at the Carnegie Institute for Science have developed the LipoGlo system ...created an endogenous ApoB-NanoLuc reporter by inserting the NanoLuc luciferase downstream of the apoB ...requires binding of bacterial enhancer binding proteins (bEBPs) at a sequence upstream of the promoter...

...053 lentiviral expression vector, which contains an sgRNA insert site and a reporter for tracing the engineered...fragment with one part of an established pair of proteins (iLID and SspB) that undergo light-induced dimerization...photosynthetic organisms use for buoyancy. These proteins allow dissolved gases to diffuse in and out but... lab discovered in 2014 that these gas vesicle proteins scatter sound waves and create ultrasound contrast...reporter plasmids, each containing two fluorescent proteins and four repeats of a miRNA target site. One fluorescent...CRISPR-Cas9 system CHIME Article contributed by Aliyah Weinstein  Listen to the CRISPR podcast segment Understanding...gene editing tool on the immune system, the group inserted sgRNAs targeting various innate and adaptive immune...

...transposon to stably integrate a reporter gene that contains a puromycin resistance gene followed by the target...the desired genome edit:  A donor plasmid that contains a puromycin resistance gene linked to an in-frame... when transfecting your cells.  A plasmid that contains Cas9. The Madison lab also created an inducible... labor-intensive, require multiple fluorescent proteins, and aren’t that efficient. To overcome these ...named GEmCherry1, due to the “genomic fragment inserted into mCherry”. However this one mutated methionine...GEmCherry2 work? The premise is that the genomic region inserted into the N-terminus of mCherry can be targeted...repaired by NHEJ which depending on the nucleotides inserted or deleted, can result in an in-frame shift of...

...While institutions that focus on creative problem solving like the Howard Hughes Medical Institute have...technology. Monoculture and chemical spraying, mainstays of modern agriculture, are now synonymous with...control over their interaction with it, we hope to inspire and open a new conversation about GMOs.  Challenge...involves two basic components: the pigments (anthocyanins), and the plant circadian clock. Petunias have...transformation technologies like those pioneered by Ewen Mullins of Ireland’s Teagasc.  Plant biotechnology is full...business enterprises, he can be found climbing mountains or playing with his two cats. Keira Havens grew...Biophysics and Biochemistry from the Illinois Institute of Technology in 2004, she accepted a commission...

...digest to cut out the inserts followed by a five-piece ligation with the four inserts and the master plasmid...mammalian or other cells. TALENs are engineered proteins that comprise a DNA binding domain and an endonuclease...technology allows you to create a pair of designer proteins that will introduce a double-strand break in the...FokI endonuclease domain as well as fluorescent proteins used to sort the cells after the plasmids have...question – cardiomyocytes, adipocytes or neurons, for instance – to see how those mutations influence the disease...particular disease state. You could use the kit to insert all of those variants into stem cells with the ...introducing a mutation somewhere else in the genome. It remains to be seen which system is cleaner in terms of ...

...constitutively expressed proteins while tet-on turns on constitutively silenced proteins. Addition of tetracycline...degron tagging vector! Destabilizing domains Destabilizing domains (DD) are akin to degron tags, just ... to temporarily reduce expression of essential proteins (when knockouts aren’t viable). siRNA or RNAi ...gene of interest. It will inhibit transcription instead of introducing a DNA double strand break. Transient...transgene vector. Degron tags Tagging relevant proteins with a degron tag can provide inducible protein... easily implemented for exogenously introduced proteins, as many of them just need a special promoter ...

...suite of plasmids (pGT) with various replicative origins that can be used for broad or narrow range of microbial...longitudinal studies of the gut microbiome, James Collins’s lab developed a paper-based method that can be...concentration within a sample. Using this system, the Collins lab was able to detect mRNA from 10 bacteria of...methods, the ability to detect mRNA transcripts of toxins and other transcripts of interest is a benefit ...with broad host-range (Meyer, 2009). The vector retains the oriT for delivery into recipients by conjugation...microbial population dynamics in the gut can provide insight into how the microbiome responds to infection, ... (DCDC), relies on the assembly of fluorescent proteins into one bright particle that segregates into ...

...enzymes that degrade RNA, are especially hardy proteins that are found in and on everything, including...solution and spun down the homogenate to remove the insoluble material. Then, the homogenate was loaded onto... hours to separate the intact RNA from DNA and proteins. Although very effective at isolating total RNA...RNA could be effectively separated from DNA and proteins by a simple extraction protocol with guanidinium...in a guanidinium thiocyanate solution. However, instead of RNA separation using cesium-chloride gradients...in the top, aqueous layer, while DNA and other proteins are retained in the interphase and bottom, organic...into one step. After homogenization in TRIzol®, insoluble material is removed via centrifugation and the...

...researchers have used fluorescently tagged DNA-binding proteins to label certain loci, but this approach is not...Cas9 (dCas9) cannot induce gene editing, but it retains its gRNA-directed targeting capacity. By using ...two hairpin sequences, which recruit fluorescent proteins BFP, GFP, and RFP fused to the respective hairpin...and red. In contrast, a gRNA with two different hairpins will produce a secondary color cyan, yellow, or... individual colors to 6. A gRNA with all three hairpins generates white light. All 6 gRNAs can be expressed...composite. gRNAs with two identical hairpin binding domains produce blue, green, or red fluorescence, depending...Learn Some of the Pitfalls When Using Fluorescent Proteins Control Protein Activity with GFP Resources on...