...less phosphatase-accessible. Kinase Translocation Reporters (KTRs) Kinase Translocation Reporters (KTRs...fluorescent kinase reporters. A KTR is comprised of a fluorescently-tagged substrate for a kinase of interest...proxy for kinase activity.  Advantages of KTRs When compared to FRET reporters for measuring kinase activity...Kinases: they regulate many proteins, with ~1/3 of human proteins predicted to be phosphorylated on at...for regulating signal transduction and measuring kinase activity at the single-cell level can aid in drawing...phenotype. One method for monitoring live single-cell kinase activity is FRET, but FRET reporters are challenging...Covert Lab provides an alternative tool with their Kinase Translocation Reporters (KTRs) whose cellular localization...

...inherited retinal degenerative disease called Retinitis Pigmentosa. I quickly became fascinated by this ...has limited tropism in the retina, primarily infecting cells of the retinal pigmented epithelium (RPE)...field of gene therapy, more specifically AAV-based retinal gene therapy. The year was 2001 and I started a...grinding stop and only now, 2 decades later, is it finally back in the limelight. On December 19, 2017 the...delivery, and they had been shown to to transduce the retina but only transiently. However new viral vectors...capable of providing long-term gene expression in retinal cells - AAV - were becoming available. At the same...AdV) and lentivirus (LV) are known to transduce retinal cells, AAV quickly became the vector of choice ...

... the second in a series of posts on bringing sustainable practices in the lab. The first post focused ...learn about some creative approaches to lab sustainability, and find ways to further reduce our consumption...consumption beyond recycling.     Choosing more sustainable products Recycling is great for rerouting waste... reused. This might sound risky in terms of contamination, but consider flow cytometers. These fluidic... grant agencies), we can shift toward a more sustainable one. The math on this is pretty simple: wash ...

...more lab sustainability tips, check out our other articles in this series: Bringing Sustainable Practices...Encourage your workplace to become a leader in sustainability. Sign up for blog posts and updates from ...individuals, companies, and universities to be sustainability leaders. Take initiative to bring ideas to ... A lot of places have small or non-existent sustainability offices, so it’s up to all of us to work on...Practices to the Lab: Innovations Bringing Sustainable Practices to the Lab: Easy Action Items References...

...have very different disease phenotypes, are two fascinating examples of epigenetics in the form of imprinting...lead to excessive eating and obesity. What is fascinating is that both syndromes can present without any... of chromosome 15. They both result from the inactivation of genes on one copy of chromosome 15 (the maternal... genes from both copies of chromosome 15 are inactivated, but different phenotypes result depending upon...suppressor genes can repress their transcription and inactivate them. Global hypomethylation, on the other hand...are known to be involved in repression and X-inactivation. The K9 reporter uses the HP1 chromodomain to...improving patients lives. References 1. Ong-Abdullah, Meilina, et al. "Loss of Karma transposon methylation underlies...

...plasmid-based and recombinant. Learn more about recombinant antibodies here. Finally, Addgene is a nonprofit...can participate: If you make recombinant antibodies or other recombinant affinity reagents, share your...the launch of our latest community resource: Recombinant Antibodies! In addition to plasmids and viral...building our antibody resource, which includes recombinant antibodies, educational materials, protocols,... 2016). It’s also been well established that recombinant antibodies are excellent for reproducibility ...and tool users. We encourage you to share your recombinant antibodies via Addgene to maximize the reach ...Additionally, many of the hybridomas from which the recombinant antibodies are derived were tested for specificity...

...Protein kinases deliver messages to the next members of their pathways, but where the kinases are located...subset of kinases. The Problem:  There is no common method for creating optically controlled kinases. The ...design for creating photoswitchable kinases and use MEK1 as a model kinase. In this system, light is used ...photoswitchable kinases: psMEK2, psRaf1, and psCDK5. MEK2 is a serine/threonine kinase with a similar ...photoswitchable kinases good for? There are a number of potential applications for photoswitchable kinases.  Below...Zhou et al. 1. Testing kinase inhibitors Zhou et al examined the ability of kinase inhibitors to alter Raf1... by intermediate messengers. Optical control of kinase activity can provide greater spatiotemporal resolution...

...high-efficiency terminators such as T0. Rho-independent termination is also known as intrinsic termination, and ...with plasmids! What are termination and polyadenylation? The role of the terminator, a sequence-based element... promoter strength as a determinant of gene expression levels, the terminator also plays an important ...degradation in prokaryotes.  Prokaryotic termination   Prokaryotic termination mechanisms fall under two general... RNA polymerase in the termination of the transcript. Rho-dependent terminators are not usually employed...plasmids use Rho-independent terminators, which include naturally occurring terminators, such as T7 and rrnB,...engineered terminators come close (>95%). For most purposes, however, any common terminator will suffice...

...channels and pumps in the membrane. An emerging interdisciplinary field of biology known as bioelectricity is...fusion of the voltage sensing domain of a Ciona intestinalis voltage sensor and a mutated version of the ...It is activated by cyan light and exhibits an inactivation maximum of 590 nm light. Aurora and Phobos optogenetic...

... but among the most important are the inverted terminal repeats (ITRs). ITRs are the elements that make...and the ITRs are retained with the gene in a recombinant AAV (rAAV) vector (Figure 1). Everything else...outside the ITRs gets left behind.    Figure 1: Recombinant AAV vector with ITRs that flank the gene cassette... Rep78 and Rep68 to initiate replication and a terminal resolution site that serves as the target site...T-shaped structure. The Rep binding element (RBE) and terminal resolution site (trs) are noted. Created with ...serotypes (Wilmott et al., 2019), and different combinations of ITR and capsid serotypes vary in their gene...plasmid DNA with mutated ITR sequences. Using recombinant-deficient bacterial strains such as NEB Stable...

...recently announced the launch of an open-access recombinant antibody resource called NABOR (Neuroscience ...The antibodies distributed through NABOR are recombinant antibodies expressed from plasmids that have ...RNA could then be used to generate functional recombinant monoclonal antibody (R-mAb) expression plasmids...preserve and share their own collections. Why Recombinant Antibodies?  One of the benefits of using a plasmid...including subclasses that were not produced by the original hybridoma. By using subclass specific secondary...Multiplex immunolabeling with subclass-switched recombinant antibodies in adult rat brain. Figure from Andrews...publicly available. The lab has also expanded their recombinant affinity reagent tools to include nanobodies....

...libraries containing combinations of different gRNAs in each plasmid. Each gRNA combination can be tracked ... an easy-to-use tool to analyze the complex combinatorial genetic networks underlying your favorite biological...screening are vastly useful for mapping the combinatorial genetics that underlie complex regulation in...barcoded gRNA libraries that can be used to combinatorially modify the genome, screen for a particular ...successfully applied to functionally characterize combinatorial gene knockouts generated via multiplexed gRNA...expression (Wong et al., 2016), in addition to the combinatorial expression of other genetic elements including...antibiotic-resistant bacteria enabled by massively parallel combinatorial genetics." Proceedings of the National Academy...

... or gene, as well as to study the effects of inactivating/activating mutations. In biomedical research.... In recombineering, bacteria express phage recombination machinery, which facilitates easy genetic modification...modification by homologous recombination. In most recombineering strategies, only 3-4 phage proteins are...a temperature-sensitive plasmid. Homologous recombination then joins the linear PCR products into stable...the recombineering plasmid is not maintained. Recombination efficiency is high enough to negate the need...own electrocompetent cells in which the phage recombination machinery is induced using L-arabinose. These... the steps of gel purification and in vitro recombination. Across all modifications described in Trehan...

... and increase your lab sustainability. For my other posts on lab sustainability, check out my Recycling...This is the last post in a blog series on sustainable practices in the lab, where I’ll list some easy...unimportant. With so many big contributors to the sustainability problem, how can one little change make a difference...quick things above, try joining or starting a sustainability project. This could be: Setting up a recycling...Learn about other creative approaches for lab sustainability Resources on Addgene.org Sign up for our ...

...maintaining intensity at ~85% of its original value after 60 minutes of illumination, highlighting its reliability...scientists. Seeing inside these specimens has offered illumination on biochemical processes that are crucial in...interpretation of FRET-based experiments. A popular combination is Alexa594, and Alexa647 due to their brightness...

...however, the other recombinase/reporter pairs were inactive, even at very high recombinase levels. These data...multiple recombinases can be used together to designate specific patterns of recombination. Finally, Rubin...rather than complete recombination. If you’re looking to use site-specific recombination in Drosophila, read... using multiple recombinases. To make multiple, precise genome edits, the recombinases used must have ...representing the specific recombination site for each enzyme, were generated to test recombination efficiency. When...showed broad recombinase-dependent RFP expression. Even at low expression levels, recombination by KD, B2...determine the cross-reactivity of these recombinases, each recombinase and reporter construct pair were tested...

...to avoid MMR: 1) E. coli with inactivated MMR: Using E. coli with inactive MMR is definitely the easier...minutes quickly inactivates the repressor and allows for expression of the recombination genes.  After ...confirmation of recombinant clones If a antibiotic resistance gene has been inserted, recombinants can first...genome engineering and is based on homologous recombination. It allows for direct modification of DNA within...called recombineering - short for homologous recombination-mediated genetic engineering.  It can be used... These enzymes then catalyze the homologous recombination of the substrate with the target DNA sequence...nucleotides of homology to the target site for recombination. Key components of the Lambda Red recombineering...

... you encounter mycoplasma contamination. Causes of mycoplasma contamination The mycoplasmas enter the ...above. The contaminants can spread through dirty clothing, lab wears, human speech near the laminar airflow... risk of contamination. Mycoplasmas can spread from these sources through cross-contamination and due ... a source of mycoplasma contamination. Due to its turbid nature, contamination is difficult to be detected...during working in the laminar airflow are also among the leading causes of contamination. These aerosols generated...cell culture process and hence eliminate one of the causes of contamination. If possible, a separate lab...bacterial and fungal contamination. Proper PPE Since humans are the major source of contamination, the prevention...

...vectors, recombination often occurs between the long-terminal repeats (LTRs). After recombination, one resulting...intramolecular DNA recombination. This naturally occurring process is mediated by recombinases like RecA and...undergo recombination, two smaller plasmids are formed. Viral vectors are prone to recombination In lentiviral...replicate.   How can I prevent recombination in my samples? Plasmid recombination can affect viral vectors ...have been engineered to further reduce recombination. Recombinase-deficient strains like Stbl2, Stbl3, ... repeating elements. Knowing how to amplify recombination-prone plasmids and testing your prep can save..., and NEB stable E. coli reduce the rate of recombination in samples. For a more detailed comparison of...

...Having never been to China myself, I was pleased to be able to participate when the organizers of the...Dmitry Samarsky, and a very welcoming team from China’s Ribobio, the illustrious speaker list attracted...winners, one NIH Director and a co-founder of BGI-China presenting, it’s no wonder it was attended by over...were potential Addgene requestors and depositors. China is in one of the top 10 countries in number of plasmid...of Guangzhou and see some of the sites. Most fascinating to me was the outdoor market where one can shop...chemistry is complex and this work requires an interdisciplinary team.  Day two of the conference was kicked...from different species for genome engineering. The final keynote presentation was by Nobel Laureate Thomas...