...undergo significant post-translational modifications including glycosylation, disulfide bond formation...proteins functionally relative to the untagged protein of interest. To avoid the accumulation of dark fluorescent...is, unfortunately, some investigators realize too late that they have missed the impact of dark, non-fluorescent... fold? Misfolded FPs do not fluoresce. A dark population is not readily apparent and can confound quantitative...construct [1,2], i.e. positioning of the FP sequence relative to cell compartment targeting sequences, which...1) [4,6–8]. Additionally, FPs that encode N-glycosylation consensus sequences are modified with the addition...addition of N-glycans [9]. Both of these posttranslational modifications can disrupt FP folding rendering...

...How are inducible promoters regulated? Inducible promoters can be regulated by positive or negative control...Addgene! Chemically inducible promoters Chemically regulated promoters are among the most common inducible ...protein purification. When arabinose is absent, regulatory protein AraC binds O and I1 sites upstream of...complexed with cAMP activator protein (CAP) can also stimulate AraC binding to I1 and I2 sites. Supplementing...include positive inducible alcohol and steroid regulated promoters commonly used in plant research. ...Example Activator Addgene Plasmid/Kit Alcohol-regulated AlcA promoter AlcR pGGA008 (AlcA) and pGGC011...AlcR) from red flame GreenGate kit Steroid-regulated LexA promoter XVE (synthetic) pFZ19 (yellow flame...

...externally. Fruit flies are also relatively straightforward to manipulate genetically. Scientists can make...make it easy to study the effects of genetic manipulations. In this blog post, we’ll cover five popular... model organism for scientists as they have a relatively short generation time for mammals - the time ...study diseases such as cancer since mice better recapitulate the complex interactions between cancer cells...are genetically tractable. Mice can be easily manipulated with tools like CRISPR to make transgenic lines...entirely sequenced and is very amenable to genetic manipulation. Yeast cells are great model organism not only...studied in other model organisms that better recapitulate in vivo conditions similar to humans. For example...

...enhance or upregulate ncRNA expression. This approach is useful for lncRNAs because they regulate nearby ...They play important roles in many cellular and regulatory processes, but much of their functionality is...unveiled as they are more difficult to functionally manipulate and study in comparison to protein-coding genes...to systems like CRISPR/Cas9 that allow them to manipulate full genes and observe the effects. Luckily for...of disrupting adjacent genes, especially those regulated by or overlapping with the lncRNA (Basak and Nithin...effects can be minimized by disrupting lncRNA regulatory elements that are crucial for transcription and...and Bester 2021). For example, inserting a polyadenylation signal downstream of the transcription start...

...antibodies bound to an ELISA plate to capture Myc-tagged sybodies. Then, the biotinylated target protein is added...coronaviruses into a llama over several days and isolated special camelid antibodies that bind to the spike...secondary nanobodies blog post.  A rapid selection platform for generating sybodies against membrane proteins...weeks (Zimmermann et al., 2020). This selection platform was designed so that any standard lab can quickly...in the form of mRNA for academic research. The template plasmids for generating your own libraries are...Generation Toolbox! Starting with libraries and a biotinylated (for purification purposes) protein target of...involves using a commercial kit for in-vitro translation of the mRNA libraries. Each mRNA molecule in ...

...gene drives” capable of spreading through wild populations. Whereas most genomic changes impose a fitness...could eventually spread through the entire wild population with unpredictable ecological effects. Because...chromosome and its own gene is used as a repair template, thereby converting the heterozygote into a homozygote...building gene drives that can spread through populations of transgenic laboratory organisms but not wild...sgRNA-only drive will spread exclusively through populations that already express Cas9 from an unlinked locus...sequence will only spread in transgenic laboratory populations with that sequence2. Both methods are easy to...performed in regions that don't have resident populations of the relevant mosquito species. Reproductive...

...expression, and protein translation. Its genome consists of “early” (E) and “late” genes, and the transition...transition between early and late infection (and therefore between early and late gene transcription) is demarcated...unusual cases of acute hepatitis in the pediatric population. Once ~400 cases had amassed in places around...exist. The life cycle of AdVs is dependent on modulation of the host cell’s cell cycle; inhibition of ...recombinant sequence (transgene) instead.    Although the late genes are usually present in the transfer plasmid...expressed, since the early genes are absent, and the late genes (and their structural proteins, which would... some of the wild-type viruses can persist as a latent infection in certain tissue reservoirs (Pereira...

...optogenetic stimulations, they simultaneously recorded the electrical impulses of discrete subpopulations of this... or reward-related behaviors. The high spatio-temporal precision and reversible modulation of FLEx vectors...combined optical and genetic methods to photo-stimulate mammalian neurons and alter synaptic events: a..., 2005; Deisseroth et al., 2006). Despite its relatively recent development, the history of optogenetics...rhodopsins and neurons have in common? Neuronal stimulations are triggered by the movement of ions across...Zhang, 2012; Towne et al., 2016). Optogenetic neuromodulation is made possible by the light: when hit with...viral-infected neurons are subsequently photo-stimulated through fiberoptic cannulas directly implanted...

...is its amenability to genetic manipulation. Tools for gene manipulation together with information about...drive biology is to examine the consequences of manipulating genes. One reason for the success of zebrafish...groundwork for understanding the gene networks that regulate processes from development to metabolism to fertility...back to) is the one-eyed pinhead mutant, which regulates a developmental pathway implicated in congenital...morpholinos complementary to a target mRNA inhibit its translation either by blocking initiation or splicing. (RNAi...(RNAi, in contrast, engages existing gene regulatory mechanisms to block activity and does not work easily...compensation, a previously unknown mechanism of gene regulation (El-Brolosy et al., 2019). Generating zebrafish...

... build the relationship in a non-awkward way?  7 ways to strengthen your new relationships Here are some...Relationships need nurturing. Perhaps you’ve met someone interesting at an event. Or, perhaps you have...contacts you have; it’s about building genuine relationships with people you care about knowing. How do you...some ways to go about strengthening a new relationship with authenticity and not discomfort: Invite the...their new publications via Pubmed, so you can congratulate them when there’s good news.  Send an email ...meeting (in person or virtually) will make the relationship stronger.  If appropriate, invite them to speak...department. Even if it doesn’t work out, it’s flattering to ask.  Show appreciation. Send a thank you ...

...customized to control transgene expression. Gene regulatory elements [promoters and enhancers, miRNA target...IV injections are typically administered in the lateral tail vein or retro-orbital sinus. IT injections... are “seeing” a great number of cells as they circulate in the blood, rather than being geographically...Abbreviations: AAV, adeno-associated virus; GRE, gene regulatory element; NHP, non-human primate. Many of...., 2023 AAV-PHP.V1* CNS: Biased toward vasculature, but also transduces astrocytes Mice Yes ...al., 2020 AAV-PHP.V2 CNS: Biased toward vasculature, but also transduces astrocytes Mice Yes ...(rats, infant green monkey, rhesus macaque); vasculature (adult marmosets), biased toward astrocytes (...

...Listen to the podcast segment on modulating iPSC reprogramming  Regulation of telomere length with TZAP .... Subscribe to the Addgene blog to get all the latest hot plasmid updates!     Tol2 Gateway-compatiable... 2015. PubMed PMID: 25772473 Targeting and manipulating interneurons using a viral approach Genetically...’s lab has developed a new AAV approach using regulatory elements that restrict expression to this cell...approach allows you to target and functionally manipulate GABA-ergic interneurons across species and will...combination of deliberate and random mutagenesis to isolate the best RFP. They targeted known residues on the...brightness, and proper maturation. Three new RFPs were isolated and characterized: mScarlet (fluorescent lifetime...

...involved “plates,” and our scientists and lab technicians always knew from context what a “plate” was. Our...these plates now” to a fellow team member. But, at Addgene, we use seven different types of “plates” (Fig...barcode for your plate, you will get in trouble if you don’t qualify what kind of “plate” you mean. Without...to know what kind of plate they were talking about during the meetings. And “plate” certainly wasn’t the...our engineers (Fig. 2). Creating this guide was relatively easy. I took a picture of virtually every container...information Prescribing an official name to all of our plates, tubes, and racks meant we had fewer errors in ...important documentation, including SOPs or project-related documents, we define new and potentially ambiguous...

...proteins that promote RNA decay and their upregulation is correlated with poor survival in breast cancer patients.... BirA* requires 16-18 hours to sufficiently biotinylate interacting proteins, which results in poor spatiotemporal...interactions with RaPID RaPID is similar to the BioID platform for identifying protein-protein interactions, ...promiscuous E. coli biotin ligase, BirA*, to biotinylate proteins that are within ~10 nm of the bait, ... also tweaked BirA* to increase its rate of biotinylation. The improved enzyme, called BASU, is derived...one to study RNAs expressed from their native regulatory elements. Controls for RaPID experiments RaPID...Affinity Purification (CRAPome), to identify highly biotinylated background proteins reported by other research...

...engineering projects in a very big way. In one of his latest papers, published in Molecular Systems Biology ...CRISPR/Cas genetic circuits are linked to the regulatory networks within E. coli host cells. ...in building genetic circuits is getting enough regulatory proteins that don’t interfere with each other...programmable that you can make a very large number of regulators. That basically means larger genetic circuits...that could conceivably be as large as natural regulatory networks. Addgene: What kinds of things could... pattern, shape. All of that is controlled by regulatory networks in the plant. If we want to access that...biology can do - it’s good to be able to build regulation thats as sophisticated as a natural network. ...

...protein’s post-translational modifications such as phosphorylation, acetylation, methylation and ubiquitination...proteins are being up- or downregulated in a system, detect post-translational modifications, quantify ...quantify protein levels relative to standards, detect the cellular location of proteins, and can be a readout...submerged in a tank filled with a transfer buffer formulated to conduct the electric current. The wet transfer...thickness of the band provides information about the relative abundance of the protein in the sample. More protein...immunosorbent assay (ELISA) (Aydin et al., 2015). Post-translational modifications In addition to protein abundance...ubiquitination. For example, to test if a protein is phosphorylated following a specific experimental condition...

...colonies on the backbone alone plate and many colonies on the backbone + insert plate (or at least more colonies...backbone alone plate). Unsuccessful ligations will usually result in few colonies on both plates (unsuccessful...), in a vector alone plate with many more colonies than the vector + insert plate (unsuccessful 2), or... each plate (unsuccessful 3).     If you have a high number of colonies on your backbone plate (greater... Follow the manufacturer’s instructions.   2. Isolate Your Insert and Vector by Gel Purification     ...throw the digestion mixtures together. You need to isolate your insert and backbone from the enzymes used ...base pairs for each varies, it is difficult to calculate this based on DNA concentration alone. One method...

... via acetylation or demethylation. Casilio DNA Methylation Editing (Casilio-ME) is a methylation editing...human pluripotent stem cells CRISPR-based DNA demethylation Optogenetic repressors of gene expression ...50–90% of average on-target DNA-editing levels relative to BE4max.  Read the paper in Nature Biotechnology...Michael Laub’s lab developed a CRISPRi system to downregulate expression of specific Caulobacter genes using...ctrA, a gene encoding an essential cell cycle regulator. The lab expressed both dCas9 and the ctrA sgRNA... demonstrated the system can also be used to downregulate multiple genes at once. Read the paper in mBio...Find the plasmids Enhanced CRISPR-based DNA demethylation Figure 2: The components of Casilio-ME1...

...Elements Translation in eukaryotes usually begins at the 5’ cap so that only a single translation event ...plasmid is packaged for viral delivery, or the relative expression levels between two or more genes is...single-stranded RNA viruses to allow for the efficient translation of multiple genes from a single transcript. The...Entry Site (IRES) to allow for initiation of translation from an internal region of the mRNA. In the ...discovered in poliovirus RNA, where it promotes translation of the viral genome in eukaryotic cells.1,2 Since...they all have in common is the ability to spark translation initiation independent of the 5’ cap.  IRES elements...bicistronic expression; however, many could be easily manipulated to express more than two genes. Plasmid Name...

... resistance genes so that you can specifically isolate bacteria containing your constructs of interest...bacteria as a way to produce more of the plasmid, isolate individual plasmids, or express a gene contained...goal of your experiment — to grow, express, or isolate plasmids. The presence of an antibiotic resistance...shaking at 37 degrees) can be shortened since the plated cells will have time to produce beta-lactamase ...to produce satellite colonies when used on agar plates due to the fact that beta-lactamase is excreted...Cons: Prone to satellite colony formation on agar plates, less stable at high temperatures and in acidic... and is less susceptible to degradation in agar plates. This prevents satellite colonies from forming ...