...destabilize the transcription complex, initiating cleavage of the transcript.   Figure 1: A predicted... monkey virus constitutive RNA transport element lead to enhanced HIV-1 Gag expression in a context-dependent...prokaryotic terminators and deposited several with Addgene (1). Prokaryotic polyadenylation Although mostly thought...initiation step of gene transcription; today we'll provide an overview on how transcription stops, or termination...from the transcription machinery. Terminators are found downstream of the gene to be transcribed, and typically...generally serves to promote transcript longevity in eukaryotes and promote transcript degradation in prokaryotes...Hepatitis Virus Posttranscriptional Regulatory Element Enhances Expression of Transgenes  Delivered by ...

...Podcast breakdown (Time : Topic) 0:00 - 1:25 : Introduction 1:25 - 6:19 : Dr. Koeris' graduate work in...Novophage's successful transformation into Sample 6 and Dr. Koeris' own transition from graduate student...37-36:53 : Was It difficult for Dr. Koeris to transition from developing therapeutics to developing a ...

... DNA that were transcriptionally active in retinal cell types in mice. Figure 1: The four strategies...used four different design strategies: Strategy #1 (ProA): Create promoters with sequences upstream of...highest rate of targeted expression. Surprisingly, <1% of synthetic promoters replicated the expression ...mouse cell type specific transcription factor binding sites interspaced with random sequences.   Strategy...synthetic promoters. Legend: TSS: transcription start site. TFBS: transcription factor binding site. Image from...particular cell type is important not only for translational research, such as gene therapy, but also basic... using naturally occurring promoters to drive transgene expression, the Roska lab built synthetic promoters...

...yourself in a creative rut, read on!   Fig 1: Blugene using creative problem-solving in the lab...Blog Transferable Skills Guide: Problem-Solving Transferable Skills Guide: Teamwork Transferable Skills...feel they lack formal training in a variety of transferable skills. At Addgene we're helping to fill this...both helping researchers recognize their MANY transferable skills learned while working in the lab and ...Today, we’re highlighting an oft-underlooked transferable skill: creativity. I’ve heard quite a few scientists..., it’s clear that creativity is absolutely a transferable skill developed during PhD training. Creativity...problem. In a study outlined in Originals by Adam Grant, a creativity expert at Stanford conducted an experiment...

...various cyan fluorescent proteins is depicted in figure 1. Of note, the photobleaching rate does not depend ...to be used with the fluorescent protein. In figure 1 we provide an example of such a comparison between...the variants is comparable (lower panel of figure 1). To avoid changes in excitation power, it is important...fluorescent proteins in the same spectral class (Cranfill et al, 2016). This non-linear dependence on excitation...the conclusions reached at high power may not translate to real applications in which far less power is...

..., including those found at Addgene! Figure 1: Strategy for assembling the barcoded gRNA library...pool-clone them into the pAWp28 storage vector (Figure 1): Forward oligo: 5’- CACCGNNNNNNNNNNNNNNNNNNNGTTTGGGTCTTCGAGAAGACCTATTCXXXXXXXXC... He can be contacted at [email protected].   References 1. Cheng, Allen A., Huiming Ding, and Timothy K. Lu....expression of other genetic elements including transcription factors (Cheng et al., 2014) and microRNAs (.... Konermann, Silvana, et al. "Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex...

...functioning T cell repertoire (1). In this example, it was possible to transduce the T-cells outside the body...significant improvements in vision over a period of 1 to 3 years – the treatment appears to be both safe...research and science communication.     References 1. Gaspar, H. B., S. Howe, and A. J. Thrasher. "Gene...their mutations corrected, and the T-cells were transferred back into the infants. Almost all patients have...techniques may hold the key. In the simplest form, translating gene therapies from conceptual design to clinical...that carries a small, approximately 4.7 kb single stranded genome. AAV belongs to the family Parvoviridae...AAV vectors are also capable of infecting a wide range of cell types including but not limited to muscle...

..., check out Addgene's CRISPR Guide. Figure 1: Like this jar of jellybeans, a pooled CRISPR library... selecting a CRISPR library for your experiments. 1.  What species are your cells derived from? Currently...a different gene. Libraries may be available in a 1-plasmid system, in which Cas9 is included on the gRNA-containing... non-specific endonuclease (Cas9) and a single stranded guide RNA (gRNA). The ~20 nucleotide targeting...of wild-type Cas9 and a gRNA generates a double-strand break in the target DNA, which, when repaired through...scaled up for genome-wide screening due to the broad range of potential target sequences and ease of generating...heterogeneous population of gRNA-containing lentiviral transfer vectors, each targeting a specific gene within...

...! References 1. Sinche, Melanie, et al. "An evidence-based evaluation of transferrable skills and job...Page Read Other Posts in the Transferable Skills Guide Developing Transferable Skills During Science Training...that they lack formal training in a variety of transferable skills. At Addgene we've set out to fill this... highlighting that researchers do learn MANY transferable skills while working in the lab and by offering...where you might need some help. Today in our transferable skills guide: Collaborating with others outside... had to overcome some hesitation to ‘approach strangers’ and learn how to communicate outside my scientific...message and to keep it short. Soon, discussing with strangers stopped feeling weird and started to be fun. I...

...Bacillus. Appl Microbiol Biotechnol. 2019 Jan;103(1):303-313. doi: 10.1007/s00253-018-9485-4. Epub 2018...and clonal analysis in Drosophila.” Nat Methods 9(1) (2011): 47-55. PubMed PMID: 22205518. PubMed Central...Methods Mol Biol. 2016;1478:53-78. doi: 10.1007/978-1-4939-6371-3_3. PMID: 27730575; PMCID: PMC5270762. ... and lac promoters.” Proc Natl Acad Sci U S A. 80(1) (1983): 21-5. PubMed PMID: 6337371. PubMed Central... polymerase and transcription factors. Since the promoter region drives transcription of a target gene...repressor then prevents transcription from occurring, which means that transcription is now OFF.   We’ll...tetracycline-controlled transactivator (tTA). They fused TetR with the transcriptional activation domain VP16...

...Zhang et al., 2015). In this system a modified TIR-1 protein can degrade degron-tagged proteins in an auxin...splitting cGAL4 in two and binding each half to a gp41-1-N-intein. Split inteins are protein domains that naturally...mutations with unc- 119 + selection." MicroPublication 1 (2018): 2–3. Wang, Han, et al. "cGAL, a temperature-robust...neurons from each class to fully characterize the transcriptome. The first data release for CeNGEN is planned... The 3’UTRs of mRNA are important for post-transcriptional regulation in C. elegans. To study the role...using a split intein for refined spatiotemporal transgene control in Caenorhabditis elegans." Proceedings...

... when (1) other strategies may not be feasible and (2) large-scale proteomic and transcriptomic datasets...expected in another. You’ll need to validate that points 1 and 2 hold true under your specific experimental conditions...application and available resources.     Figure 1: Table summarizing the IWGAV five proposed antibody...validation: A view from the mountains. New Biotechnol 45:1–8. https://doi.org/10.1016/j.nbt.2018.08.002  Uhlen...Interest (GOI), and transfect it into your favorite cell line. In parallel, you transfect cells with another...antigen - Recognition of an immunogen does not guarantee recognition of the target antigen. You want to...Antibody-independent methods may include analysis of transcriptomic or proteomic data from several different tissues...

...and P. mirabilis, had only ~1 transfer per 1,000,000 colonies. Higher transfer rates are better suited for...non-selective plate. Most bacteria tested had 1 transfer in every ~100 to 1,000 colonies, but some bacteria...active for greater than 50 generations. Figure 1: Mobile-CRISPRi's modular components. Image from Peters...protocols can transfer CRISPRi into a single bacterial strain, such as a B. subtilis, or a narrow range of bacterial...can be introduced into a broader range of bacteria. Once transferred, Mobile-CRISPRi stably integrates...bacterial Tn7 transposon system. This system has two parts: a plasmid expressing the Tn7 transposition genes ...chromosomal copy of the RP4 transfer machinery mobilize these plasmids for transfer to recipient cells during...

... (which range between 0 and 100) are then converted to a weight (which ranges between 0 and 1) so that...values (ranging from -40 bp to 40 bp) are also converted to a weight (which ranges between 0 and 1) (shown...distance) This gives each guide a score ranging between 0 and 1. The cutting frequency determination (CFD...summary per batch. Ranking guides for HDR in-depth In other tools, guides are ranked by specificity of ...genome by eye. We came up with a ranking formula that does just this: Rank = (compressed CFD score) * (gaussian...interactions (e.g., scores >80) are transformed to 100. This transformation (illustrated in the graph below...employs biological best practices. For example, it ranks guides by CFD score, and it mutates HDR donor templates...

...activating kinase 1 (MEK1) with either one or two phosphoserine residues cotranslationally inserted in their...directionally assembled into a Level 1 vector creating a single transcriptional unit (Ex: a promoter, 5’UTR, ...expression. Brown et al., Genesis 2014 Jan;52(1):39-48. APEX2 for proteomic mapping and electron...engineering cells that lack release factor one (RF-1; Bacterial strain EcAR7) and minimizing the set of...comprised of three sets of cloning vectors (Level 0, 1, or 2) which can be utilized in three successive assembly...coding region, and terminator). Next, up to six Level 1 modules can be similarly assembled into a Level 2 ...vectors to allow for additional iterations of Level 1 assembly if necessary. Combining multiple Level 2 ...

...Please see Figure 1 for commonly used markers for major mammalian organelles and transport pathways. It is...cell with the images from the Allen Cell Explorer (1). When studying the function of a protein or its role...Subcellular Localization Poster here! References 1. Allen Institute Plasmid Pages 2. Cooper GM. 2000....can be gained by understanding the location and transport of the protein within a living cell. Analyzing...Analyzing differences in protein localization and transport between healthy and diseased states can also provide...endoplasmid reticulum, modified in the golgi and then transported in vesicles via the secretory pathway to their...the end of their life cycle, proteins may be transported through the vesicles to the lysosome where they...

...homologies. Abbreviated protocol for PITCh Step 1: Generate microhomologies in the PITCh vector ~20...editing possibilities MMEJ can enable. References 1. Sakuma, Tatsushi, et al. (2016).  “MMEJ-assisted ...CRISPR-Cas9 with the PITCh systems.” Nat Protoc. 11(1): 118-33. PMID: 26678082. Find the plasmids from ...genome edits. But have you heard of another double-stranded break repair mechanism: MMEJ (microhomology-mediated...primary methods for repairing DNA after a double-stranded break. HDR copies the sequence from a repair template...are chewed back to reveal homology, allowing the strands to anneal. DNA synthesis then fills in the gaps...5-25 bp) regions of homology flanking a double-stranded break yield precise deletion of the sequence between...

...biological questions. The two most common uses are: 1) to test the expression level in a specific system...brightness is compared to that of EGFP which is set as 1. Some proteins are very dim (e.g. TagRFP657, which...Photostability can be as short as 100ms (EBFP) or as long as 1 hour (mAmetrine1.2). However, for most FPs it is a...or Förster] resonance energy transfer). FRET occurs when energy transfer from one FP (e.g. CFP) excites...classified by the emission color (emission wavelength range) FPs are usually classified by emission color as...as outlined below (or emission wavelenght range).  By mutating GFP, the variants blue FP (BFP), cyan FP...nm Green 499 - 519 nm Yellow 524 - 538 nm Orange 559 - 572 nm Red 574 - 610 nm Far-Red 625 -...

... products). I picked this post for a few reasons: 1) I think phage are super cool and have considered ...Connect the Research Community Grad School Advice Part 1: Picking a Lab and a Project That’s it for now. As...Editor's Choice Blog Posts As I’m sitting in the San Francisco International Airport listening to the Lion King...back from a very successful Outreach Trip in San Francisco and would like to give my thanks to all the Addgene...

... spread out onto the plate. We test the (capped) 1:1 agarose/DMEM solution against a part of the body ...a 60 mm dish ready for picking is shown in figure 1.   Find Additional Useful Protocols on Addgene's Protocol...growth rate/transfection efficiency is not known, this is easily achieved by splitting transfected cells into...specific cell line, and its transfection efficiency. We often trypsinize the transfected cells on the day after...after transfection into plates containing selection agent at different densities, then dispose of the plates...under-expressing clones. If GFP-encoding cDNA is co-transfected at a favorable ratio to the gene of interest ...such as the Viaflo from Integra Biosciences) to transition from a 48-well cell plate to a 96-well assay ...

...nucleotides are represented equally, that’s only a 1/16 chance of having a PAM at a given site. While it... NYN/NRN NYN is less effective than NRN Table 1 - Summary of PAM-flexible Cas enzymes Are we truly...editable with CRISPR technology.     Fig. 1: SpCas9 has sparse PAM coverage in the human genome.... The activity of SpRY was validated in double stranded cleavage and base editing applications, with high...solution? Almost! Assuming a PAM of NRN, 50% of randomly selected genomic loci will have a PAM located ...

...Listen to the interview with Darrell Kotton Chapter 1: Introduction to Darrell Kotton and the Boston University...Unit, the Pulmonary Consultation Service, the Alpha-1 Center, and the Pulmonary outpatient clinic, all at...Chapter 3: How all basic research ends up being translational. Ex: the discovery of GFP. Chapter 4: Goals ...

...backbones for shRNA expression, such as pLKO.1 puro and pLKO.1 – TRC cloning vector. Lentiviral Plasmids ...rapidly in 2011, from zinc finger nucleases to transcription activator-like effector nucleases (TALENs). ...

... for less money. The key steps to colony PCR are: 1) design primers to detect the presence of your insert...primers. There are 3 strategies for primer design: 1) insert-specific primers, 2) backbone-specific primers...previously described. When using insert-specific primers (1), positive clones (+) will give a band, while a negative...control. A good positive control is bacteria transformed with the same backbone plasmid. If this control...strain. A good negative control strain is an untransformed culture of the same strain of bacteria you used...Cloning Technique Learn to Perform a Bacterial Transformation Deposit Your Positive Clones with Us ...

...determined LY6A, also known as stem cell antigen-1 (SCA-1), is the receptor used by PHP.B to reach the CNS...William L. Stanford. "Concise review: stem cell antigen‐1: expression, function, and enigma." Stem cells 25.6...PHP.B has a >40-fold increase in CNS transduction and transduces the majority of astrocytes and neurons... mice whose CNS was transduced by PHP.B but absent in mice who were not transduced by PHP.B. The Deverman...These results were surprising because in vivo transduction of AAV serotypes generally tracks between strains...diverse library of AAV9 mutants that had a small randomized peptide loop inserted in the AAV9 capsid. This... an enhanced version of PHP.B, shows similar transduction efficiency, but at a log lower dose than PHP.B...

...individual activities (1). Researchers also discovered the modular format of some transcriptional activators: that...requirement for transmembrane proteins, a split ubiquitin system has been devised (8). References 1. Casadaban...systems could be combined effectively (3). In Figure 1 for instance, the DBD of the fusion protein shown ...flurry of discovery in the field of eukaryotic transcriptional activation and cell biology. During this period... that the DNA binding domain (DBD) and transcriptional activation (TA) domains retain their individual... regardless of whether it interacts with a transcriptional activator (1a) or a repressor (1b). Scientists...interactions. In later years, improvements to yeast transformation protocols also helped establish the yeast two-hybrid...

...tethered to bring them into close proximity (Figure 1). Formation of the complete FP results in a fluorescent...detected by fluorescence microscopy. Figure 1: How BiFC works. A split FP (e.g., yellow fluorescent... protein interactions in living cells. Nat Protoc 1:1278–1286. https://doi.org/10.1038/nprot.2006.201 ...Forster Resonance Energy Transfer (FRET). Through the non-radiative transfer for energy between neighboring...Whether BiFC constructs are expressed transiently by transfection or stably impacts the success of BiFC..., kinase signaling, integrin signaling, and transcription factor interactions (Kerppola, 2006). One of...interactions between basic leucine zipper and Rel transcription factors (Hu et al., 2002). This would be more...

...microscopic resolution for studying colloidal gold (1).  The method was based on using a thin plane (sheet...Siedentopf, and R,. Zsigmondy.  (1902) Ann. Phys.  315, 1 – 39.
 A. H. Voie, D. H. Burns, F. A. Spelman. (1993...microscopy/en_de/products/imaging-systems/lightsheet-z-1.html ...microscopy coupled with techniques that make tissues transparent are enabling microscopes to visualize the cellular...the cortico spinal tract, CST, spinal cord and cranial nerve afferents labelled with a AAV8 expressing...sectioning (OPFOS) (2). Arne Voie, David Burns and Francis Spelman focused a laser beam into a thin sheet ...of clear specimens and for in vivo imaging of transparent organisms. Advances in tissue clearing Similar...

...centrifugation (lenti) Centrifugation Titration time ~1 day (AAV); ~1 week (lenti) 2–10 days Initial titers Higher...will help you decide which is right for you. Table 1: Mammalian versus insect cells for viral production...only have to worry about cloning your transfer plasmid. The transfer plasmids have different packaging capacities...AAV and lentivirus requires three plasmids — a transfer plasmid containing your gene of interest plus ...in mammalian cells is relatively quick. After transfection, HEK-293T cells start producing virus within...systems, it is standard to do only one passage (transfection). This differs from typical insect cell preparations...an additional couple of days, as it requires transduction of more cells, while AAV can be titrated following...

...Strong mammalian promoter from human elongation factor 1 alpha CAG Constitutive Strong hybrid mammalian promoter... sense or coding strand of the transcribed gene. The coding strand is the DNA strand that encodes codons...to the mRNA transcript produced. The antisense strand is referred to as the template strand or non-coding...non-coding strand as this is the strand that is transcribed by the RNA polymerase. DNA sequences called response...initiation of transcription whereas transcription factors promote the initiation of transcription. The most...template for the translation of a protein. RNA polymerase III transcribes genes encoding transfer RNAs (tRNA...reference material covering promoters, transcription and the transcription complex. Plasmid...

...acceptable. Aims should be mechanistic, doable by 1-3 people in 1-5 years, and contribute to resolving the overarching...possible, do at least two full length practices (~1 h) and get feedback. It’s also helpful to practice...aims do not need to be grant aims. Each aim could be the basis of a single grant. Either way, think realistically...talk could be your plans for your first NIH R01 grant proposal or a 10-year research plan. I suggest preparing...is a significant need for healthy livers for transplants. Growth of new livers in culture could resolve..., neither of these can substitute for a liver transplant. My lab will combine bioengineered scaffolds ... for presenting a science talk Check out our transferable skills guide Read all of our career blog posts...

...experiments or even negatively impact cells (see Figure 1). When behaving properly, FPs enable researchers ... disulfide bonds in the secretory pathway (Figure 1) [4,6–8]. Additionally, FPs that encode N-glycosylation... sarcoma-associated herpesvirus.       References 1. Snapp, Erik. "Design and use of fluorescent fusion...excellent choice for fusions with integral membrane proteins and membrane associated proteins (i.e. GPI-anchored...these compartments undergo significant post-translational modifications including glycosylation, disulfide... addition of N-glycans [9]. Both of these posttranslational modifications can disrupt FP folding rendering...including the secretory pathway (Figure 2), the inner membrane space of mitochondria and chloroplasts, the extracellular...

...environments such as live cells and tissues (Figure 1). These flexible strategies give you the best of both...majority of in-cell labeling strategies have two parts: (1) a genetically encoded “tag” expressed as a fusion...Follow him on twitter @rhodamine110.   References 1. Liu, Zhe, Luke D. Lavis, and Eric Betzig. "Imaging...requires more photons. For example, moving from transient overexpression of protein fusions to gene-edited... lipoic acid ligase, biotin ligase, phosphopantetheinyltransferase) – These enzymes catalyze the covalent... tag (Figure 1c). Click chemistry (e.g., transcyclooctene–tetrazine)– Nonnatural amino acids can be ...improved brightness, photostability, and spectral range. Nevertheless, these fluorophores were designed ...

...a rate of up to 1 per 500-1000 bp. In comparison, the DNA replication error rate is 1 per 109 bp. Unlike... and Screening via Transcriptional Output-TANGO (PRESTO-TANGO) to screen the NCC-1 library of approved...(MAGE) (1). MAGE uses recombineering (2) to simultaneously incorporate multiple single-strand DNA (ssDNA...generated 5 common 30bp overlap sequences (Sites #1-5). Each overlap site encodes a unique restriction...First, GMAP-compatible backbones for lentivirus LV 1-5 and retrovirus RV 2-5 were constructed. The authors...of GFP (SEP) by a 9 amino-acid linker (see Figure 1). The key to the biosensor lies in pH: DsRed is relatively...inactivate SEP and leave only DsRed to fluoresce (1). With variants that can be targeted to specific cellular...

...’re not one of these people yourself.  Fig 1: Quote from Michael Ende, courtesy of morefamousquotes.com... blog Management for ScientistsTransferable Skills Guide: LeadershipTransferable Skills Guide: Teamwork...the right time. Like many of the posts in our Transferable Skills series, this post is not meant to be ... that you could strengthen. The right set of transferable skills, coupled with your technical skills, ... can help, and if and when you find yourself transferring this skill to another job, it will be important...also aggravate long-term personal conflicts.  Transitioning a conflict into a collaboration may not be the...willing and able to solve, don’t take that skill for granted. When you go on interviews or are looking for a...

...my master’s, I worked in an academic lab where I ran a handful of assays over and over. Everyday was the...help you decide if a master’s might be for you. 1. Are you interested in master’s-level jobs? Ultimately...your work at conferences, help with manuscript and grant preparation, and lead your own research project....

.... pH and DNases affect DNA stability Figure 1: So many choices! What to do? The main factors ...your downstream experiment. TE (10 mM Tris-HCl,1 mM EDTA, pH 8.0) buffer is the best buffer for preserving...from the DNA. These apurinic sites if in double stranded DNA are repaired by base excision repair, but ...but can lead to mutations in single stranded DNA undergoing replication.   DNase on the other hand can digest...

... Figure 1: With blue light, the VVD domains of the BLADE dimerize and activates transcription. The ... that dimerizes upon exposure to blue light (Fig. 1). Another blue light-inducible dimerization domain...from one that represses transcription to one that activates transcription.However, this system lacks spatio-temporal...barbara_synbio) May 13, 2021 The original AraC transcriptional regulator Their system is based on the AraC...AraC protein that controls transcription from the PBAD promoter. “AraC is widely used in synthetic biology...It relies on the sugar arabinose to activate transcription by causing conformation changes in the AraC ...your gene of interest, all you have to do is co-transform pBLADEONLY. That way, a previously cloned gene...

... genetic tools in Drosophila (Fig. 1):  GAL4-UAS, a transcription factor binding site. GAL4 expressed...Rodríguez et al., 2011).  Genetics of SPARC Figure 1: SPARC and SPARC2 use the GAL4-UAS system along with...visualize individual/non-overlapping cells. While transgenic animals are commonly used to specifically manipulate... to trigger recombination and expression of a transgene (Lin et al., 2018). However, this system requires...site to cleave and inactivate any read-through transcripts of the effector gene. Effector genes, any gene...across cell types and organisms by replacing the transcription factor binding site and effector in the construct... the SPARC system to use other experimental transcription activation systems, such as LexA/p65 or mCD8...

...our lives. Plasmid transformation into E. coli is a fairly inefficient process– just 1 out of 10,000 cells...down especially fast and plates should be used witin 1 month for optimal efficiency. Beware of satellite ...aminoglycoside Binds 30S ribosomal subunit; causes mis-translation Bactericidal 50-100 ug/mL Spectinomycin aminoglycoside...Blocks 50S ribosomal subunit; inhibits aminoacyl translocation Bacteriostatic 50-100 ug/mL in EtOH Polymyxin...Polymyxin B polypeptide Alters outer membrane permeability Bactericidal 10-100 ug/mL Tetracycline tetracyclin...Binds 50S ribosomal subunit; inhibits peptidyl translocation Bacteriostatic 5-25 ug/mL in EtOH *In prokaryotes...