...Neveu, 2015). Origin and purpose of MXS chaining MXS-chaining was designed to create plasmids for fluorescence... principle behind MXS-chaining is repeatable, restriction-enzyme based (chaining-based) cloning. The modules...promoters Gibson et al., 2009 MXS-Chaining, BioBricks, Bglbricks, etc. Chaining-based methods (restriction enzyme-based...similarly apply MXS chaining to your experimental needs. Let us know how you use MXS Chaining by emailing us....org. Table 3: Chaining Based Cloning Methods Method Restriction Enzymes Chaining Direction MXS-Chaining...useful application of high-throughput cloning. MXS Chaining is one such technique and has been used to produce...example, modular constructs generated with MXS-chaining were used to label subcellular structures in live...

... stocks and use of the original Cell line you purchased, keeping all experiments carried out on low passage...you to track a cell line’s age and monitor any changes. Your tracking system could be as simple as a piece... stocks to work from. The future Attitudes are changing but yet more is needed to convince people of some...Lacks. New York: Crown Publishers, 2010. 2. Gold, Michael. "A conspiracy of cells." State University of New...

...tools, producing lentivirus, can pose certain challenges. Whether choosing a system that is the best fit...will provide an overview of some of the common challenges associated with producing and using lentivirus...make these optimization experiments a bit more challenging. With these considerations in mind, when planning...viral components; according the manufacturer these changes result in yields up to 25x higher than other available...such as Cas9. See figure 1 for an example of how changing transfection ratios can affect transfection efficiency...

...testing and a lack of well-characterized genetic tools make it challenging to engineer a specific function...areas with our color-changing flower, but to do that we’ve had to navigate the challenges involved in plant...Additional work has also identified and characterized mechanisms and components of the circadian rhythm...excited to see how this exchange of genetic tools facilitates plant science. Challenge #3: Intellectual property...fibers. And because working with plants can be challenging, there are a lot of unexplored areas in plant...plant synthetic biology. Challenge #1: Public perception of synthetic biology One of the biggest difficulties...manner. At Revolution Bio, we are addressing this challenge directly by developing a plant biotechnology product...

...created SRIRACCHA in 2017 to enrich and identify desired CRISPR mutants (Wen et al., 2018). SRIRACCHA uses...Madison lab also created an inducible SRIRACCHA system (iSRIRACCHA) where Cas9 expression is activated ...these reporters, editing typically results in a change in the fluorescent readout. In this blog we will...focus on two fluorescent CRISPR reporter methods, SRIRACCHA and GEMCherry2. We will cover single base-pair...editing CRISPR reporters in the near future.  SRIRACCHA (stable, but reversible, integrated reporter for...genome of mammalian cells.  Figure 1: The SRIRACCHA reporter construct. Image from Wen et al., 2017...for easy screening of different gRNAs by simply changing the gRNA used when transfecting your cells.  A...

...  Quantitative polymerase chain reaction, qPCR Quantitative polymerase chain reaction (qPCR) also known...What is polymerase chain reaction? If you have ever worked in a molecular biology laboratory you have...have likely done a polymerase chain reaction (PCR). PCR is an in vitro method in which a small amount of...thus a heat resistant enzyme that can withstand changes in temperature. Taq was first identified in the...standard curve, which can be time consuming and challenging to get right. ddPCR uses a water-oil emulsion...Quantifying low abundance targets Monitoring subtle changes in target DNA levels Multiplex PCR Amplify... Resources on Addgene.org Protocol: Polymerase Chain Reaction (PCR) Protocol: How to Design a primer  ...

...Shen, and Michael Z. Lin. "Optical control of cell signaling by single-chain photoswitchable kinases."... Figure 1: Photoswitchable MEK1 (psMEK1). Is it possible to create other photoswitchable kinases? The...in the Michael Davidson Collection Find Photoswitchable Fluorescent Proteins ... lab present a generic design for creating photoswitchable kinases and use MEK1 as a model kinase. In ... MEK1 activity by attaching two dimerizing photoswitchable Dronpa (dron= a ninja term for vanishing, and...site of a constitutively active MEK1. This photoswitchable MEK1 is called psMEK1. When exposed to 500 ...psMEK1 also worked for creating three other photoswitchable kinases: psMEK2, psRaf1, and psCDK5. MEK2 is...

...Continued development of new allelic exchange vectors Allelic exchange is a great technique for the scarless...allele exchange in bacteria." Plasmid 51.3 (2004): 246-255. PubMed PMID: 15109831. Pyne, Michael E., et...modification. This technique is frequently called allelic exchange. Here, a mutated allele introduced from a conjugative...off-target mutations. Ultimately, successful allelic exchange requires two sequential homologous recombination...counterselection vector is the pDS132 sacB-based allelic exchange vector. This vector contains sacB, an chloramphenicol... colonies isolated are those where the allelic exchange vector has undergone single crossover and integrated...escape). The rhamnose induction system is well-characterized and quite tight (Giacalone et al., 2006). The...

...going virtual for Addgene’s first AAV Data Hub Challenge, now taking place Dec. 6, 2022! Share your AAV...

...that this basic concept of gRNA design is still challenging for researchers using CRISPR, and that standardizing...paraphrased in the tweet below. The complexity and challenges of homology directed repair (HDR) are echoed ...s a list of blog posts to help you tackle the challenges of DNA repair (with bonus gRNA design resources... DNA for Enhanced CRISPR Genome Editing Chris Richardson and members of Jacob Corn’s lab describe how ...a brand new CRISPOR manual to kick off 2018! "Mechanisms of precise genome editing using oligonucleotide... donors". Miura, Hiromi, Rolen M. Quadros, Channabasavaiah B. Gurumurthy, and Masato Ohtsuka. Nat Protoc...Creating knock-in mouse models with CRISPR has been challenging, with protocols using dsDNA templates achieving...

...Streak Plate Challenge, Westenberg’s course includes Winogradsky columns and characterizing the microbes...plate technique. Time for the #BioSci3319 #StreakPlateChallenge part 1. Lab B needs a redo :-( @SandTBioSci... the lab used three different species for the challenge, but one species always outcompeted the other ...it would be a perfect fit for the streak plate challenge. Chromoproteins are a subset of the fluorescent... distinguish colonies from one another in the challenge. To create a successful streak plate, first the...colonies in the last quadrant after incubation. The challenge is usually successful for the students. “They ...colonies. I should probably make it a bit more challenging, potentially by using a thicker bacterial suspension...

...thanks to Alex Chavez for taking the time to sit down and talk with us.  Alejandro (Alex) Chavez is a member...episode of the Addgene podcast, we sit down with Alex Chavez, a Clinical Fellow in Pathology at Masachusetts...Learn More About Alex's Work with Cas9 Activators Michael Koeris Explains His Path from Grad Student to Entrepeneur...

...Additional Career Posts on the Addgene Blog How did this change in labor laws come about? In March 2014, the White...Institutions are now preparing to comply with the changes to the law on Dec 1st, according to guidance issued...stipend levels. In theory, therefore, the salary changes expected for postdocs in many cases should be as...to gauge exactly how postdoctoral salaries are changing. Transparent salary information for postdoctoral...policies are one indicatator of how salaries may changes due to the FLSA. In 2014, 89% of institutions had...salary range. We have been able to gather data on changes to salary for some of the institutions that are...what they are doing, however, may face greater challenges. Current salary information that we have been...

...I include in my chalk talk?" So what exactly is a chalk talk? On the surface, a chalk talk is quite simple...present a "chalk talk." However, few, if any, faculty job candidates have seen an actual chalk talk. Their...first exposure to a chalk talk is usually their own. This is a problem. The chalk talk is effectively ... hone a chalk talk. Yet, I often hear, "I have an interview next week and it includes a chalk talk. What...steps to chalk talk preparation: Define your research vision. Organize your vision into a chalk talk format...simple. It's you, a dry erase board, some markers (chalk is rare these days), and a room of 5-30 faculty ...format. Practice. Define your research vision A chalk talk could be your plans for your first NIH R01 ...

...optogenetics.  Challenges of engineering channelrhodopsins There are three main challenges to engineering...machine-learning to overcome channelrhodopsin design challenges To overcome these challenges, the Arnold and Gradinaru...the expression of light-gated ion channels such as channelrhodopsins (ChRs) makes them light-responsive... shines on excitatory channelrhodopsin-expressing neurons, the channelrhodopsins respond by opening and...deliver channelrhodopsins to the brain. This delivery route concentrates lots of channelrhodopsins in one...since current channelrhodopsins tools have low conductance, but also confines channelrhodopsin activation...conductance compared to current channelrhodopsins. Their new channelrhodopsins, called ChRgers, make the brain...

...variable light chain (VL) and variable heavy chain (VH) of an antibody. These two chains are connected ...developed hybridoma technology. But what about single chain fragment variables (scFvs)? scFvs weren’t developed... for additional rounds of panning and scFv characterization.    Figure 2: Phage display starts ...plasmid in bacteria (Ahmed et al., 2012). One challenge of bacterial expression is the proper formation...cytoplasm. expression in the presence of molecular chaperones that help scFvs fold properly in the cytoplasm...Lee T, Pope S, Riordan G, Whitlow M (1988) Single-chain antigen-binding proteins. Science 242:423–426 . ... of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. Proceedings...

...cold conditions. However, many of the detailed mechanisms that allow extremophiles to survive under these...fruit juice clarification, meat tendering, and oligosaccharide synthesis. A major incentive to use cold-active...folding at low temperatures, since mesophilic chaperones would not function well under suboptimal conditions...engineering a plasmid which contained cold tolerant chaperones (the Cpn 10/60 isolated from Oleispira antarctica...the figure below shows, E. coli expressing the chaperones protein resulted in higher levels of growth compared...discussions. Not only are such endeavors a welcome changeover from conventional academic settings, these are...

...dedication to resource sharing has led us to develop a searchable and sortable datatable that contains validated...and dCas9-FokI. This table is both sortable and searchable, which can help exclude the data that you are... numeric columns.  The data in any column are searchable and you can search by any part of the data. For...

...in cancer. Yet, the majority of them remain uncharacterized. This represents a considerable barrier to ...Their work culminated in the first functional characterization of over 1,000 mutations, and the group has...are also sensitivity issues when attempting to characterize mutations in a pooled cell population, as mutations... Michael G. Lemieux...

...plasmids is available in an easily searchable format on Addgene’s Michael Davidson Fluorescent Protein Collection...great work of Michael Davidson and his colleagues." Acknowledgments Thank you to Michael Davidson, Michelle...proteins and their use for imaging cell biology, Michael Davidson’s lab at Florida State University has ... the cell, use these markers as your guide. As Michael Davidson told the Tallahassee Democrat in a story...

...installment in our podcast series, we chat with new Addgene Board Member, Michael Koeris. Dr. Koeris did his graduate... capital firm, Flagship Ventures, Koeris helped change Novophage's focus. The company went from developing...pathogens in food products. Along the way, the company changed its name from Novophage to Sample 6. To learn ...

...excited to announce the recipients of this year’s Michael Davidson and Roger Tsien Commemorative Conference...conference of their choice while honoring the late Michael Davidson and Roger Tsien who have played enormous...fluorescent protein tools. This year, we’ve made some changes. In addition to supporting in-person conference...health and disease,” says Aggarwal. This biosensor changes fluorescence based on the release of glutamate ...colleagues Filip Tomaska and Tim Hanson, are characterizing variants of iGluSnFR3 in vivo and have used...be transferred to the roGFP2 cysteine thiols and change its fluorescence properties. The biosensor can ...allow us to better understand how neuron activity changes following opioid withdrawal, providing insight ...

...Susanna Bachle Anion-conducting channelrhodopsins (ChR) are ion channels that can be used to silence the...PubMed PMID: 30166441. MerMAIDs - marine channelrhodopsins for the optogenetic toolbox Article contributed...

...Listen to the photoswitchable protein podcast segment Reversible control of photoswitchable proteins, which...the light- and dark- states of the photoswitchable cPYP, which changes in conformation in response to blue...picking approach to select cells with variant characteristics they wanted, the team identified Archon1, a... Discovery of new protein binder tools for photoswitchable proteins cPYP and AsLOV2 Article contributed... which change in protein conformation in response to light, offers a way to spatially and temporally control...structural knowledge of the proteins. To overcome this challenge, the Uppalapati and Woolley lab developed a phage...bind to only the light- or dark- state of the photoswitchable proteins offers a powerful approach to control...

...Young-Gie Chung, Mi-Yoon Chang, Baek-Soo Han, Sanghyeok Ko, Eungi Yang, Kwang Yul Cha, Robert Lanza, and Kwang-Soo...Plasmids for Stem Cell Research Delivery Method Mechanism Efficiency Safety Pros Cons MMLV-derived Retrovirus...element that transposes via a “cut and paste” mechanism between a vector and chromosomal DNA. PB has a...be removed by re-expressing the transposase. A challenge of using PB to generate iPSCs is that genomic ...the cytoplasm independent of the cell cycle. A challenge of using Sendai is that since it’s replication...clones tested had plasmid integration. Another challenge is that multiple transfection makes it difficult...plasmids due to lower activation of DNA-silencing mechanisms. While minicircles are typically smaller than...

...activity, channelrhodopsin-2 (ChR2), a blue light-activated cation channel, from the archaebacteria Chlamydomonas...light, this channel activates neuronal activity by opening and allowing positively charged ions to rapidly.... Light-gated ion channels directly open and close when exposed to light and change the membrane potential...receptor or pathway.  Light-gated ion channels Light activated ion channels (rhodopsins) either excite or inhibit... range of wavelengths. Excitatory channels allow positively charged ions such as Na+, K+, and Ca++ to ...Tichy et al., 2019), photoswitchable tethered ligands (PTL), and photoswitchable, orthogonal, remotely-tethered...with each light source used.   Channel and activation kinetics Some channels turn on and off very rapidly...

...particularly new. Chapter 2, “The Professional Scientist,” addresses the challenges of succeeding in science...Do-It-Yourself Science” described in Chapter 3, and of course the rapidly changing world of genome engineering ...call bad science.”  The three chapters in this section describe the challenges of trusting science in a “...science’s biggest challenges over the coming years. It is arguably the most subjective chapter of the book,...to. The problems and challenges described in The State of Science have not changed in “COVID times,” but...scientists to change their priorities. For many scientists, COVID-19 will fundamentally change what they ... that may have warranted their own chapters, and certainly chapters that could warrant their own books...

...committee, I became Vice-Chair when the current Vice-Chair stepped down. When the Chair then stepped down, ... your own development as a leader. Chairing a committee I was Chair of the Advocacy Committee at the NPA...member. The NPA runs its committees through Chairs and Vice-Chairs that lead them. As a committee member, ...the other members on the team and keep the Chair and Vice Chair of the committee updated with their progress...became interim Chair automatically as it was an understood expectation of any Vice-Chair. Following an ...there were many changes to the  Board of Directors and the Executive Director changed twice. I had to ...Leadership Institute” (CLI) where each Committee Chair and Vice Chairs were invited to spend a weekend together...

...real-time, posing a huge challenge to studying epigenetic changes over time.  This challenge was difficult to ...the study of heritable changes in gene expression that do not involve any changes to DNA sequence (Epigenetics...modifications can result in phenotypic changes without any change in genotype. Epigenetic modifications... within an organism. Mechanisms of epigenetic modifications The main mechanism of epigenetic modification...DNA is wrapped around histones changes, then gene expression can change as well. The more frequently and...epigenetic changes were statistically significantly associated with reduced life-span, but no changes in the.... Chromatin remodeling can occur by two main mechanisms, either through histone modifications, which ...

...chloride channel SwiChr++ - A channelrhodopsin converted into a chloride-conducting anion channel. It is...the role of ion channels and pumps in developmental biology.  Examples of some ion-channel and ion-pump ...ChR2 (blue-light activated cation channel) light-activated channels to hyperpolarize tumor cells, which...BLINK2 potassium channel - An optimized blue-light induced synthetic potassium channel. Upon activation... can be measured as the imbalance of electrical charge between the inside and the outside of the cell....cell. This imbalance is maintained by ion channels and pumps in the membrane. An emerging interdisciplinary...role of bioelectrical signaling mediated by ion-channels and pumps in developmental biology, regeneration...

...when a B cell changes which heavy chain constant domain it pairs with its variable chain domain. B cells...of an antibody labeled with Fc, Fab, heavy chain, light chain, constant region, and variable region. ...in their heavy chain constant regions result in several unique physical characteristics of these five ...by the heavy-chain constant genes μ and ẟ. These are the first two genes in the heavy-chain constant domain...isotype? An isotype determines several key characteristics of an antibody as well as the role it plays...class of antibody that’s determined by its heavy-chain constant region (see Antibodies 101: Introduction...antibody isotypes that each have a unique heavy-chain constant region: IgM, IgD, IgG, IgE, and IgA.    ...

...annurev-neuro-111020-100834 Challis, R.C., Ravindra Kumar, S., Chan, K.Y., Challis, C., Beadle, K., Jang, ...spiral ganglion neurons Mice, rats No (mechanism unknown) Chan et al., 2017 Asencor et al., 2022 ... Deep Parallel Characterization of AAV Tropism and AAV-Mediated Transcriptional Changes via Single-Cell...10.1016/j.ymthe.2019.02.013 Huang, Q., Chan, K. Y., Tobey, I. G., Chan, Y. A., Poterba, T., Boutros, C. L....or lumbar spine. Image and caption courtesy of Challis et al., 2022. Where do they come from? Systemic... (>1 x 1012 vg total in mice) may increase the chances of inducing an immune response against the capsids... capsid in your strain of interest, check the mechanism of entry if known, and, as always, validate the...

...composed of four peptide chains (two identical heavy chains and two identical light chains) each with a constant... between one heavy chain and one light chain. When assembled, the four chains make a Y-shaped protein ... The chains are covalently linked to one another by disulfide bonds between the two heavy chains and between...comprise the entire light chain and a variable and constant region of the heavy chain. As the name implies,...= Variable domain. L = Light chain. C = Constant domain. H = Heavy chain. Created with biorender.com   ...Fab’ fragments are almost interchangeable, but the extra bit of heavy chain on a Fab’ can act as a convenient...portions of the constant regions of the two heavy chains. In the lab, this is the region that determines...

...building block for further chaining. The MXS-chaining kit contains 5 empty chaining vectors plus a destination...large changes in binding affinity with light stimulation. Indeed oLID showed only a two-fold change in affinity...binding assays, to engineer photoswitchable dimers which show over a 50 fold change in binding affinity with...with MXS-chaining. Analogous to modular assembly methods, such as BioBricks, the MXS-chaining method uses...are also ready for further chaining. Plasmids constructed using MXS-chaining were used in a variety of ...order to determine the relationships between pH changes and cellular events. To overcome the issues with...(BRET) were developed by the Johnson Lab . Characteristics of the pHlash reporter protein include: H+...

.... Congratulations to Paula Videira, Michael McMurray, and Richard Gronostajski who each are being awarded...27393411. Michael McMurray: Rescuing arginine mutations in rare disease with guanidine Michael McMurray...PMID: 9514755. Richard Gronostajski: NFI-related diseases of neural development Richard Gronostajski is...manifestations. PMM2-CDG is the most frequent CDG type characterized by deficient phosphomannomutase (PMM) activity...suffer from immunological defects. These are characterized by a predisposition to severe and recurrent ...surprising. However, the molecular and cellular mechanisms behind the immune defects in CDG patients are...The primary focus of his lab’s research is the mechanisms by which proteins assemble into functional multi-subunit...

...many have mechanisms in place to prevent the burden from getting overwhelming. These mechanisms include ...Cases present specific operational or commercial challenges or questions that clients face imminently. Consulting...on analyzing the situation that presented the challenge at hand and help resolve it through well-informed...this realm, there are a few different kinds of challenges that consultants get called upon to tackle, depending...to collaborate whenever it faces an important challenge. The types of projects mentioned above are just...rewarding parts of the job is being confronted with challenges that are crucial for the success of a drug’s ...reasons why client situations might be especially challenging. For instance, the client might need to get a...

... looking for a flow antibody, the YCharOS (Antibody Characterization through Open Science) initiative ... community produces antibody characterisation data as part of the YCharOS open science ecosystem. It has...initiative has now started producing flow cytometry characterization data for selected antibodies, using the knockout...antibody selectivity. Additionally, it can be a real challenge to troubleshoot RNAi failures. Potential explanations...cell survival or proliferation, an additional challenge is that only partial or ephemeral knockdown will...confirmation of antibody selectivity even more challenging. Correlation with RNA or Proteomic data from ... control containing treated cells. The expected change in expression should then be reflected in antibody...

...Cascade-Cas3 mechanism. Created with BioRender.com.   The reeling mechanism The reeling mechanism is carried... DNA in a number of ways, from small edits that change single base pairs, to chromosomal inversions and...light. One lesser-known Cas is waiting for its chance in the spotlight — Cas3. Cas3 can induce genomic...recruit Cas3, which carries out the brunt of the mechanical work for editing. Class 1 CRISPR systems make...of CRISPR-based immune systems in bacteria and archaea. Class 1 is further broken down into different ... contain Cas6. Regardless of the subtype, the mechanism of Cascade binding and subsequent DNA degradation...this complex, developing Cas3 tools has been challenging. It can be difficult to ensure comparable expression...

...to look at the other channels you’re using first. A different fluorophore channel may be affecting your... be challenging! But we hope this guide can help you get started. Remember, you can always change your...process of correcting spillover from one fluorescent channel to another. When you label your samples with multiple...similar wavelengths (See this handy emission spectra chart to find your antibody's spectra). Your cytometer...population. By compensating between fluorescent channels, you can correct for this spillover and more easily...using a tool like BioLegend’s Fluorophore Guide Chart.  Pro tip! Some antibodies go by different names...look like and how do I fix it? When plotting two channels against each other, you should be able to draw...

...abundant, charged tRNAs but will stall at regions calling for low abundance, poorly charged tRNAs. When...different codons, modify the tRNAs correctly, and charge the tRNAs with the appropriate amino acids. If ...of codons, it only needs to produce a subset of charged tRNAs and therefore may need fewer resources for...encoded by codons with low abundance or poorly charged tRNAs may be produced at a lower rate than proteins...proteins encoded by highly abundant, charged tRNAs. For example Tuller et al. found that translation efficiency... by a mixture of codons with highly and poorly charged tRNAs, different regions of the protein may be ... this may give the swiftly translated regions a chance to fold properly. For example Pechmann and Frydman...