...dTomato) that promote interactions between the FPs interfere with native interactions, preventing proper ...the red fluorescent proteins form tetramers. In general, fluorescent proteins have a natural affinity and...higher order aggregates. This property can be tolerated in some applications (e.g. labeling of cells or...homodimers or higher order oligomers can be analyzed by several techniques. Gel filtration and SDS-PAGE under non-denaturing...reflect the likelihood that fluorescent proteins interact or oligomerize in cells? This question is largely...fluorescent protein’s propensity to homodimerize. Several studies have used the OSER assay to demonstrate...

...specific to the acceptor In order for FRET to occur several constraints must be met. The donor and acceptor...appropriately for energy transfer via dipole-dipole interaction. The efficiency of FRET can be measured for a...FRET range, FRET efficiency is used to infer an interaction between the fluorophores and serves as a small-scale...As the choice of fluorophore pairs is a key consideration for a FRET experiment, the possibilities can...of fluorescent proteins available. Fortunately, several excellent reviews have collated data from various... typically used for studying protein-protein interactions by measuring the change in FRET efficiency, ..., FRET experiments can probe protein-protein interactions, measure the concentration or activity of small...

...Performance dCas9-VP64 activation is generally thought of as the “first generation” CRISPR activator. While it ...initially published in 2013 (Bikard et al., 2013, Perez-Pinera et al., 2013). In the years that followed, innovative...Performance SunTag performs better than first generation activators while showing lower activation levels...to recruit transcription factors. Performance Generally, VPR is found to have significantly higher activation...method, dCas9 is fused to CBP, an histone acetyltransferase domain capable of rearranging chromatin structure...588 . https://doi.org/10.1038/nature14136 Perez-Pinera P, Kocak DD, Vockley CM, Adler AF, Kabadi AM, Polstein...

...your study is aimed at developing human therapies. If a therapeutic is the end goal, it is best to work with...Targeting is also important in the development of therapies when expression must be restricted to specific... crossing the blood-brain barrier.  Another consideration when picking ligands for chemogenetic studies...ligands. However, they retain their intracellular interactions and still set off the same pathways, giving ... (Saloman, JL, et al., 2016).   DREADD ligands Several DREADDs, including hM3D(Gq), have been derived ...& Gold, M. S. (2016). Gi-DREADD Expression in Peripheral Nerves Produces Ligand-Dependent Analgesia, as...

... In this article we will discuss general viral safety considerations and best practices for handling lab-grown... basic biological research as well as clinical therapy. These biological tools are useful because they... separate plasmid (envelope plasmid), and the polymerase (Pol) for replication and Gag structural protein... and the same holds true for types of viruses generated in the lab. The four most common types of viruses... lab and require additional safety measures.   General viral handling safety   Containment and personal...resistant to ethanol and isopropyl alcohol, 0.5% peracetic acid, 10% bleach, or 1% iodine are useful. Some...

...circuits with functional relevance following a set hierarchy. MoClo originally developed from Golden Gate Cloning...organization schema MoClo can be broken down into three hierarchical levels: individual DNA parts, transcriptional...For example: in a cell there are proteins which interact with other proteins and with DNA, and these complexes...are multigene systems whose protein products can interact with the DNA that encodes the protein itself or... or other genes within the circuit. These interactions yield a cellular phenotype, translational output...can support a larger cellular engineering goal, several of which are described below. Multigene circuits...promoter for a given peptide can easily be tested by generating Level 1 TUs with unique promoters, and the ideal...

... Overview of the CRISPR classification hierarchy. The hierarchy flows from Class to type to subtype to...the array of CRISPR technologies. Learning the general principles of this classification system will help...classifications were placed into a fairly simple hierarchy: class to type to subtype to variant. Despite ...seemingly complicated steps to arrive at this hierarchy, the main aspects of each class and type are easy...Class 1, and are the most common type of CRISPR overall. Type I systems degrade large sections of DNA thanks...most common. Figure 4: Overview of the general CRISPR type VI mechanism. The Cas13 effector binds...diversity of rare CRISPR-Cas systems with deep terascale clustering. Science, 382(6673). https://doi.org...

...cascades that clear these infections. Anything that generates an antibody response in the immune system is referred...churn out antibodies against the injected protein. Several weeks later, blood from the animal is drawn and...antigen binding, the variable regions alone can be generated as a fusion protein. An ScFv is made up of the...your samples to a standard curve. This curve is generated using a serial dilution of a known amount of protein...for other experiments to study protein-protein interactions. Flow cytometry Flow cytometry is used to detect...2014.   In vivo uses For both research and therapeutic purposes, antibodies can be used to affect cellular...functional grade antibodies. Some of the research and therapeutic uses overlap: antibodies can bind to and inhibit...

... virus may acquire mutations to escape these therapeutics. Jesse Bloom’s lab at the Fred Hutchinson Cancer... Figure 1. (A) SARS-CoV-2 RBD DMS Library generation and sequencing. From Starr et al Cell., 2020....synthetase complex, the team investigated whether the generated antibodies could bind these aaRSs. They found ... Gräslund lab plans to continue their work by generating antibodies for additional aminoacyl tRNA synthetases...nanoparticle can protect proteins from unfavorable interactions in the cell, improving biosynthetic pathway ...represent 0.001% of the population.  Because indel generation by Cas9 can occur at a steady rate, scientists... Farm GENIE project have arrived! We now offer several versions of GCaMP8 vectors in serotypes AAV1 and...

...Evan’s team generated their Myc antibodies. At first, anti-c-Myc [9E10] was just one of several new tools...over the years, several alternative antibodies against the Myc tag have been generated in an attempt to.... However, it was (and still is) infeasible to generate highly specific antibodies for every potential...would help the field explore Myc’s role in cell proliferation and tumorigenesis.  Using two different synthetic...epitope within that region. You’ll notice that “generate a reliable antibody for protein tagging approaches...

...carbon sources such as glycerol or glucose (in moderation) may also serve to increase cell density and ...plasmid yield. You can optimize your growth time, temperature, and shaking speed to maximize cell density and... and plasmid yield as these factors are generally, but not perfectly, correlated. Typical growth times...sufficient gas exchange in the overnight culture. Generally, large cultures in flasks may be shaken around...plates, require a faster shaking speed for proper aeration (260-300 RPM). 37℃ doesn’t work for every plasmid...plasmid Certain plasmids and strains grow best at temperatures other than the standard 37℃. This plasmid feature...recommendations to grow a plasmid at 30℃ or room temperature. These recommendations also often require a longer...

...describe and interact with the world using words like OR and AND. These logical operators are so useful...collection is the beginnings of what we think of as an operating system for engineering an entire cell. Typically...BIOFAB project is seeding the beginnings of an operating system for programming life, starting with the... pieces in these kits as the beginnings of an operating system or language for programming life. Which...as applied to products on the domestic market generates $300 billion dollars every year. That's two percent...anticipate plugging into the probiotic and cellular therapeutic space. If you were to look forward to the future...

...be stabilized by monomeric superfolded GFP and averages only ~50% RNA knockdown. In initial tests, Cas13b...Prevotella sep. P5-125 (PspCas13b) yielded 62.9% average knockdown, and they chose this enzyme for further...editing. To increase A->G editing, they employed hyperactive ADAR constructs, like ADAR2DD(E488Q). They also...and designated this system REPAIRv1. Using next-generation sequencing, they confirmed A->I editing and found...off-target activity. By mutating residues in ADAR2 that interact with duplex RNA, they hoped to destabilize ADAR-RNA...   Addressing the potential use of RESCUE in therapeutic applications, the authors tested various dRanCas13b...of RNA editing in research and its potential therapeutic uses We are also curious if REPAIR and RESCUE...

... to illuminate biology and enable next-generation therapeutics. Prime editing, base editing, PACE, and... lab places an order, they are likely to order several plasmids at once, breaking the statistical independence...our distribution, resulting in the following hierarchical model: Because the U.S., Europe, and China ...the inferred percentage of normal activity (the average rate of unique PIs/labs placing orders per day)...The inferred weekend effect is, in retrospect, generally more dramatic than the COVID-19 effect, though...not a causal interpretation -- the effect would generally not be the same if weekends were somehow imposed...

...fresh columns, regenerated columns had similar DNA yields. Additionally, columns regenerated up to five times...size of bacterial culture you’re working with. Overall, the glass syringe filter method is not only cheaper...cellulose dipstick. Image: Zou et al., 2018. Regeneration of silica columns for DNA purification Starting... and Deng labs developed a simple method for regenerating silica columns that come with PCR product purification...before a wash with ddH2O and then ethanol. After regeneration, trace amounts of DNA (3 femtograms/ul) could...Huang, L., Zhao, S., & Deng, W. (2018). Rapid Regeneration and Reuse of Silica Columns from PCR Purification...

...company that is developing RNAi therapeutics to treat neurodegenerative diseases. He has earned a B.S. ...s Board of Directors, Bart Newland. Newland is General Counsel at Atalanta, an early-stage biotech company...like? BN: I've made the transition now to being general counsel of a small start-up. For the last 20 years...and Biogen. They’re very different jobs. Being general counsel at a small start-up is very interesting...interesting. It’s very varied. There’s IP work to do, general corporate work, and negotiating contracts with other...

...opportunity to discover and evolve your transferable skills. Transferable skills are the non-technical skills... 5 o’clock. You’re going to have to talk and interact with...non-scientists! Maybe you’ve just spent... Wu in graphic design. You could dread these interactions, but you could also embrace them. Talking about...production, I didn’t really start to recognize my transferable (or “soft”) skills until I was out of the lab...lab to think about these--get started with our Transferable Skills Guide.     Over the last decade, my... blog Visit our careers topic page Read our transferable skills guide Read blog posts about career readiness...

... slim chance of generating indels with base editors, keep in mind that the generation of indel sometimes...minimizes the second outcome and increases the generation of the desired T-A base pair. Figure 1...inosine), which is treated as ‘G’ by cellular polymerases. Subsequently, an original genomic A-T base pair...simultaneously.     These binary vectors could be used to generate stable genome edited plants via Agrobacterium-...transformation. Cytosine base editors for plant There are several CBEs available for plant systems. Target-AID based... transient assay. For stable transformation, regenerated plants may be subjected to genotyping. If successful...

...means they can be used with or as fluorophores in several different ways. Researchers have used oligonucleotides...can be created in many different ways, but the general premise is that an aptamer in its unbound state...cells. The new aptamer also has a higher melting temperature than its Spinach ancestors, and has a lower magnesium...fluorophores, including: Environments with higher temperatures, since proteins can denature while aptamers ...likely to trigger immunoresponses. Nucleotides, in general, are far less immunogenic than proteins. Assays...ions or particularly small molecules that do not generate an immune response. Aptamers can be selected for...Peixuan AND Haque, Farzin. RNA Nanotechnology and Therapeutics. Boca Raton: CRC Press, 2014. 5. Hayat, Akhtar...

...base-pair mCherry insertion. Editing of these motifs generates lesions that are subject to excision and ssDNA...This edit changes the histidine to a tyrosine generating a GFP variant (GFPY66) that has a shifted emission...Reporters in Gene On include a mScarlet reporter, luciferase reporter, and neomycin resistance reporter. Image...hypothesis with GFP fluorescence. To do this they generated a silent GFP construct, that contains an ACG start... of different reporters including mScarlet-I, luciferase, antibiotic resistance markers, and other enzymes...fluorescent proteins, antibiotic resistance, and luciferase. References Gaudelli NM, Komor AC, Rees HA,...CRISPR/Cas9-mediated Genome Editing. Molecular Therapy - Nucleic Acids 5:e334 . https://doi.org/10.1038...

... the ASM General Meeting (now Microbe) in 2013 in Denver, CO. While I had been to the General Meeting ...the next generation of scientists. The connection between mentors and Singletons can accelerate the networking...meet someone new, as they’d been introduced to several people by their mentor, as well as their bin-mates...recognized by meeting organizers in their town hall.  Iterative improvement - Making Binning Singletons better...Goldschmidt conference, Coastal & Estuarine Research Federation (CERF) Biennial Conference, Gordon Research Conference...

...process to reduce viral order fulfilment from an average of 40 minutes to 10 minutes. While users can’t ...result in less than optimal user experience and the overall soundness of the software design. Each of us brings...called Scrum. The Scrum framework is based on an iterative and collaborative approach to development. Scrum...capacity to finish all of the work. Since we work in iterative chunks, we have the flexibility to adapt and easily...allows for life to happen without impacting the overall work. Each of our sprints is themed and the stories... being maintained (i.e. hasn’t been updated in several years) or they make significant changes that make...that helps our users focus on the fun stuff, accelerating research and discovery. We work with transparency...

...your viral vector, you use the same protocol for generating lentivirus, but you use a different envelope ...terminals and can’t be used for neuronal tracing or peripheral gene delivery. Combining the rabies glycoprotein...Cells Are Inactivated by Human Serum. Molecular Therapy 2:218–222 . https://doi.org/10.1006/mthe.2000.0116...Modification of Hematopoietic Cells for Research and Gene Therapy. Viruses 12:1016 . https://doi.org/10.3390/v12091016...Lentiviral Vectors: One Vector, Many Guises. Human Gene Therapy Methods 28:291–301 . https://doi.org/10.1089/hgtb...transport and access to the nervous system after peripheral delivery. Human Molecular Genetics 10:2109–2121...envelope glycoprotein show increased stability in sera and augmented transduction of primary lymphocytes...

...continuous evolution and protein engineering to generate seven new prime editors, PE6a–g, that overcome...delivery and reduce off-target effects of gene therapy, Feng Zhang’s lab has developed a system called...specific cell types. It can also make CAR-T cell therapy more accessible by simplifying the production process...creation of CAR-T cells directly within the body. Overall, DIRECTED can increase on-target delivery with ... offers a promising approach for experimental therapies and medical applications.  Find DIRECTED plasmids...Lacy Proximity labeling can identify protein interactions or map a local proteome, but some applications...transcriptional history of a cell. Lin et al. generated a fusion protein called iPAK4 that assembles into...

...and some considerations when using the method. ELISA basics All ELISAs follow the same overall process....simple and saves time, it also has several drawbacks. Direct ELISAs generally have higher levels of background... the capture reagent to prevent non-specific interactions. Next comes the binding step where the target...Typically, the reporter is an enzyme such as horseradish peroxidase (HRP) or alkaline phosphatase (AP)... of conjugated antibodies   Additional considerations No matter what ELISA method you choose, it ...

...initiation proteins to start their own replication. In general, these plasmids tend to be low copy number. Conversely...at the low-affinity sites, the proteins work cooperatively to separate nearby AT-rich regions of DNA. In...are occupied by ATP-bound DnaA, the proteins cooperatively can separate the DNA strands. Created with BioRender.com... from the ori. In the elongation phase, a DNA polymerase synthesizes a new DNA strand at each replication...controlling the initiation of replication. Once the average copy number for a given plasmid has been reached...called iterons located near the ori and directly interacts with DnaA, stabilizing its binding and contributing...negatively regulate its own expression and thereby the overall replication of pSC101 (del Solar et al., 1998)....

...India at room temperature. Partnering with our distributor allowed us to ensure that temperature-sensitive...transit for a certain number of days and tracking average transit time for each courier we contracted with...destination in a timely manner and at the correct temperature. To date, we have partnered with six distributors...country. ‌For example, when we began offering temperature-sensitive materials, we noticed a large percentage...preps and libraries were kept at the appropriate temperature for the duration of their journey. Figure 2... backbones, lentiviral packaging systems, and luciferase expression. On individual material pages, we ...

...molecule into the cell. The fluorescence that is generated allows for qualitative and quantitative measurement...contact with its RNA aptamer. This increased interaction between Corn and its small molecule makes Corn...developed to quantify RNA transcription by RNA Polymerase III. By expressing Corn under promoters for transcripts... transcripts uniquely transcribed by RNA Polymerase III (5S, tRNA and U6) and measuring Corn fluorescence...effect of mTOR inhibition on the activity of RNA Polymerase III. Red and orange fluorescent aptamers Corn...changes in intracellular viral rna." Journal of General Virology 98.5 (2017): 992-1003. PubMed PMID: 28555544...PMC5602116. Song, Wenjiao, et al. "Imaging RNA polymerase III transcription using a photostable RNA–fluorophore...

...these techniques are cumbersome, often requiring several rounds of selection to isolate plants with the ...organisms like Arabidopsis and Medicago truncatula and several crop species including potato, corn, tomato, wheat... plant cells.   This blog post will present a general overview of plant genome engineering using CRISPR... repress target genes in plants using the same general experimental design principles developed in other...systems, multiple gRNAs can be expressed to modify several genomic loci at once (get more information on multiplexing...sequence to enhance targeting to the nucleus, and several codon optimized Cas9 variants have been created... the plasmids described above, Addgene carries several useful CRISPR toolkits for creating plant expression...

...of organelles and vesicles, and more. Although several strategies are available to disrupt microtubules...extended to other GTPases. The Chow Lab has also generated a variety of membrane-recruitment optogenetic ...recently developed a full toolbox and workflow for generating new Rho GTPase optogenetic systems (Berlew et...express the other separately, then use blue light to generate CRY2-CIB1 oligomers that trap the fusion protein..., and 100 times the activity of the original, generated using directed evolution (Tei et al., 2023).  ...license.   Just make sure you don’t overdo it! The moderately-enhanced 10x and 30x SuperPLDs avoid dark-state...Krishnan, N., Ononiwu, F., Pal, D., Manikas, J., Taveras, C., Hall, N. A., Silva, J. D., Freshour, J., &...