...the proteins cooperatively can separate the DNA strands. Created with BioRender.com. Once DnaA breaks...it recruits helicases to continue unwinding the strands. This creates two replication forks that move in...elongation phase, a DNA polymerase synthesizes a new DNA strand at each replication fork. Termination sequences...act as a feedback mechanism to reduce further transcription. Replication is also regulated by the balance... concentration is controlled at the level of translation. RepZ mRNA must fold into a “pseudoknot” held...intramolecular base pairs for the protein to be translated. An antisense RNA called Inc, also encoded by...prevents the mRNA from folding and blocks RepZ translation (Asano & Mizobuchi, 1998). This system keeps ...

...fluor. Transcribing and translating a protein fluorophore also takes time - bypassing translation altogether... could be screened for their affinity to a wide range of non-nucleotide targets. These RNA molecules, ...The key to aptamers’ flexibility is their broad range of targets, including proteins, peptides, amino ...ions, and cells (even pathogenic bacteria). That range is possible because aptamers can either fold to ...biosensors. Aptabeacons Fluorescence resonance energy transfer (FRET) technology has been studied and used in...fluorescence-activated cell sorting (FACS) of E. coli transformed with aptamer libraries, the Jaffrey group selected...

...Hola! Que tal? My name is Brandon Solis, and I am a rising senior studying Sociology and Dance at Skidmore...interests are very clear, I had no idea how to translate them into real world careers. I figured this internship...a way of challenging what I am used to. Brandon Solis, marketing intern. Something that I struggle...world, I was worried about how my ideas would be translated through Zoom calls and Google docs. I pondered... do was create content that was unique but also brand friendly. I introduced the team to a new design ...program, Canva, and I was able to create cute and on-brand templates for these posts. Some of recent ... Brandon Solis...

...scientists, tech transfer offices, research foundations, and funding initiatives such as Fast Grants that support...worked closely with the legal partners and Tech Transfer Offices at universities to make COVID-19 research-related...United States, and many universities and Technology Transfer Offices were, at least, temporarily closing, these...Texas at Austin University of California, San Francisco  University of Pennsylvania Stanford University...them available to industry, please reach out at techtransfer@addgene.org and we will initiate a conversation...conversation with you and your technology transfer office to discuss the possibilities for expanded sharing. ...

... tissues are nearly transparent. As Verkhusha explained it to me, the transparency window of mammalian...tumor. They wanted to find something better. Transparency window That’s exactly what they reported doing...proteins that would fall within that near-infrared range, and that’s exactly what iRFPs achieved. Those proteins...corals, but all of them fluoresce outside the transparency window of mammalian tissues,” Verkhusha said...near-infrared fluorescent proteins within this transparency window, so now, we could see deeper.” Adding... proteins that operate within the 750 to 800 nm range, and his team is also looking in the direction of...

...emphatically deny that I have a magic formula to guarantee a reader the faculty job of his/her dreams. There...currency of scientific success is publications and grants. 2. Write succinct and easy to understand documents...years. You will discuss the focus of your first grant, how you will overcome certain obstacles, alternative...sentences) What will be the focus of your first grant? (1-2 sentences) Simply stated, what are your...It's not about you and how you will finally be granted the faculty position you have craved since grad...how YOU will fit into a department and bring in grants. What kind of colleague will you be? Do you understand...takes to be successful in running a lab, getting grants, and writing papers? This means that once you get...

...is composed of single stranded DNA, which is particularly well suited for strand invasion, a key step ... are then packaged into AAV virions via transient transfection of 293T cells with helpers, and used to...non-sequence specific frameshift mutations through aberrant double strand break repair, it has proven more challenging...identical targeting vectors delivered through transfection of naked DNA. Other investigators who had been...AAV-based gene editing via the introduction of a double strand break by using a targeted nuclease. Asuri et al...

...dopamine (DA), a well-known reward chemical and neurotransmitter. But aside from DA’s famous role as the “happy...standard method of measuring DA and other neurotransmitters in the brain has been in vivo microdialysis...performed with a probe surrounded by a semipermeable membrane, which is inserted into the region of the brain...passive diffusion of DA across the semipermeable membrane from the extracellular fluid into the perfused... to a threatening looming stimulus in vivo in transgenic zebrafish Report DA release in live mice over...developments in the detection and measurement of neurotransmitters in various species and while exhibiting complex...Dopamine-sensitive adenyl cyclase: possible role in synaptic transmission." Science 174.4016 (1971): 1346-1349. PubMed...

...bound to anything? Is it a transmembrane protein, or is it know to translocate or fold in certain conditions...as part of performing a specific function. A transmembrane protein that is being used as a marker for flow...cell, instead of when it is attached to a cell membrane.  Picking the Right Antibody  It can get even ...looking for a protein of interest while it’s in a transition state or specific configuration. So how do you...

...reverse transcribe their gRNA into DNA, which is then transcribed into mRNA before being translated into ...genome types that can be either double stranded (ds) or single stranded (ss) (ie, dsDNA, ssDNA, dsRNA, positive... of virus. RNA genomes can be transcribed into mRNA, directly translated into proteins, or, in the case...

...factors that could be transferred between cells. At the time, these extranuclear agents of heredity were...was proposed a few years later by Élie Jacob and François Wollman and became the widely adopted name for...-factors. Like F-factors, R-factors could be transferred between bacteria via cell-to-cell contact; however...pSC102, with EcoRI and selected for E. coli transformants that were resistant to both. When this proved...Chizuko Ogata, and Sachiko Sato. “EPISOME-MEDIATED TRANSFER OF DRUG RESISTANCE IN ENTEROBACTERIACEAE VIII:...PubMed Central PMCID: PMC314833. Datta, Naomi. “Transmissible Drug Resistance in an Epidemic Strain of Salmonella...

...field is a series of fluorescence resonance energy transfer (FRET)-based calcium biosensors. Perhaps most ...developed and published in 2010 for visualizing the transcription factor cAMP-responsive element-binding protein...really important and when? That is not known.” Transition Metals Maarten Merkx of Technische Universiteit...particularly for the intracellular imaging of transition metal ions, such as zinc, copper, and iron. “...the sensors involves plenty of trial and error. Transition metals also present challenges in that they are... way to make a FRET sensor with a large dynamic range,” he says. Merkx recently applied this “trick” to...red-shifted FRET sensor for zinc using variants of mOrange2 and mCherry as donor and acceptor domains. He ...

...contamination from uncut plasmid. 4. Transformation Transform your ligation reaction into your bacterial...perform, at minimum, two transformations after a ligation: 1. Control Transformation containing the ligation... how many colonies you should expect in the transformation due to background re-circularization and contamination...competent cells. For most standard cloning, you can transform 1-2μl of your ligation reaction into competent...ligation mixture with backbone alone; 2. Transformation containing ligation mixture with insert and backbone...conduct a positive control to ensure that your transformation worked. You should also verify that you are...

... seems straightforward: E. coli bacteria are transformed with the library DNA, they grow and replicate... Each Addgene library has its own depositor transformation and amplification protocol, but the overall...Maintain representation of the library during transformation First, use enough DNA and competent bacteria...and we use electroporation to ensure a good transformation efficiency. For a typical library, Addgene ...growing the cells on solid plates, where each transformant forms its own colony, uninfluenced by the growth...concern, but Addgene still recommends plating the transformation mixture on solid media. The colonies are then...that were amplified preferentially). You can also rank the plasmids by abundance, scaled to 1, to create...

...more detailed level, the Gal4/UAS system is a transcription activation system co-opted from yeast (1). It...workhorse to turn genes on or off. Gal4 is a transcriptional activator that binds to UAS enhancer sequences...sequences found in DNA. It then recruits transcription machinery to the site to induce gene expression. Thus...reads “#” as a “pound symbol”. References 1. A. H. Brand, N. Perrimon, Targeted gene expression as a means...15262411. 4. T. D. Southall, D. A. Elliott, A. H. Brand, The GAL4 System : A Versatile Toolkit for Gene ...Lai, T. Lee, Genetic mosaic with dual binary transcriptional systems in Drosophila., Nat. Neurosci. 9, 703...The Q system: a repressible binary system for transgene expression, lineage tracing, and mosaic analysis...

...responses are varied; one outcome is directed transcription from a given promoter. To harness the power ...the RR (OmpR) via phosphorylation, promoting transcription (see figure below). Subsequent exposure to red...response regulator via phosphorylation, promoting transcription of a GFP reporter. Subsequent exposure to red...activity persisted in the inactive state. The dynamic range, defined as the difference between the lowest and... reduced system leakiness and increased dynamic range to around 100-fold. These improvements will allow...Applications of these systems The large dynamic range of these systems ensures that they can be used with...types of proteins, including Cas9 and dCas9-transcription factors, for which low expression levels are...

...Wondering how you can develop more transferable skills? See Developing Transferable Skills During Science Training...of the University of British Columbia. When transitioning from an academic science career path to a non-academic...advantage over other candidates. Write about your transferable skills Some of the skills you developed might...research. For example, did you help put together a grant application? Are you familiar with the publishing...have presented in some public lectures? These transferable skills could be meaningful for your potential...Originally on track for a research career, she transitioned into a communications role instead after she...

...codon, which is then transfected into the desired cells. A hybrid mRNA transcript containing luciferase...luciferase, usually serves as an indicator of transcription within cells, where detection of the reporter...Higher Lower Background Lower Higher Post-translational Modification Not Required Required Photobleaching...regulatory elements, such as promoters, enhancers and untranslated regions, or the effect of mutations of these...as the most efficient means to disrupt the cell membrane and deliver the substrate; however, secreted luciferase...luciferase and the gene of interest permits translation of functional luciferase, until miRNA target and degrade...

...synaptic transmission. Cleavage of VAMP2 renders it inactive, thus inhibiting neurotransmitter release....Plasmids podcast Light-mediated block of synaptic transmission Article contributed by Klaus Wanisch  Listen...enable VAMP2 cleavage, and suppresses neuronal transmission within minutes. Constructs using the photodimerizer...utility of light-mediated suppression of synaptic transmission in brain slices from mouse, and showed that ...recognized as important regulators of post-transcriptional gene expression across the majority of cellular...an unregulated fashion and thus a marker for transfection. The other (mKate2) is regulated in the 3’ UTR...

.... elegans. These dsRNAs target specific mRNA transcripts for degradation. Despite how easy these experiments...often completely ineffective or results in low penetrant phenotypes that are hard to study. To overcome...target gene between two inverted T7 promoters. Transcription from each promoter progresses past the specific...podcast segment A fundamental approach to studying membranes, organelles, and other subcellular compartments...enzymes function across a much broader temperature range than BioID, which only functions optimally at 37...and C. elegans.   Find the TurboID plasmids here! Branon et al. Nat Biotechnol. 2018 PubMed PMID: 30125270...This tetramer cleaves chlorophenol red-b-D-galactopyranoside on the assay paper turning it purple, allowing...

...brightness When a dish of mammalian cells is transiently transfected with a plasmid containing a fluorescent...fluorescent proteins, e.g. see Chudakov et al (2010), Cranfill et al (2016) or Thorn (2017). However, the theoretical...developed in our lab uses a reference protein that is translated from the same open-reading frame and separated...reference protein) under identical conditions, a ranking of the practical brightness can be made (Goedhart...repeating this with another fluorescent protein, a ranking of practical brightness can be made. This strategy...the comparison of the practical brightness of two orange fluorescent proteins, differing by only a single...

...the filament from (a-b), showing the appearance and disappearance of 649-mDia1 or 549-CP (upper, middle...takes is a bit of chemistry magic to produce a wide range of useful molecules linked to that substrate. If... with SiR-CA (magenta) and outer mitochondrial membrane labeled with SNAP-OMP25 with Atto590-BG (green...separately with different dyes. b) mDia1 and CP appearance at the filament tip from (a). c) Intensity traces... probes are not very good at crossing the cell membrane. So, you should investigate your options and weigh...are typically 230–250 aa. Smaller size isn’t a guarantee that a tag won’t disrupt the function of the target...https://doi.org/10.1038/nmeth749 Strebinger, D., Frangieh, C. J., Friedrich, M. J., Faure, G., Macrae, R...

...Cas9 fused to the reverse transcriptase (RT), which induces two double-strand breaks and deletes the intervening...They further develop a version of SEND for gene transfer with pseudotyped viral particles, expanding options...enzyme from the Liu lab to target opposite DNA strands and specify a large intervening deletion. Not only...deletions and introduce epitope tags and RNA transcription start sites. The lab further demonstrated that...uses paired pegRNAs targeting complementary DNA strands to program large genomic deletions with or without...PEDAR is capable of correcting larger genomic rearrangements as demonstrated in a mouse model of tyrosinemia...

... has recently published techniques to follow translation in living single cells. However, before CRISPR-Cas9...the Max Planck Institute for Brain Research in Frankfurt am Main, Germany. She presented a novel technology...to be invited to learn from them at the MPI in Frankfurt am Main and perform similar experiments. Visualizing...Both antibodies have a short antibody specific DNA strand added to them. Only if the proteins of interest...are in close proximity to each other can the DNA strands take part in rolling circle DNA amplification. ...microscope she likes to travel, discover new restaurants, go to concerts and bake. you can contact her...neurons reveals a mechanism for regulating its translatability." Science 343.6169 (2014): 419-422. PubMed ...

...their mutations corrected, and the T-cells were transferred back into the infants. Almost all patients have...repertoire (1). In this example, it was possible to transduce the T-cells outside the body and thereby correct...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...disease-associated mutations. AAV vectors maintain persistent transgene expression over many years in postmitotic, long-lived...targeting and enhances endonuclease delivery for double-strand break repair." Gene therapy 17.9 (2010): 1175-1180...

... efficient double stranded templates, rather than the more efficient single-stranded oligodeoxynucleotide...generally very small, and can contain different neurotransmitters. Using genetically encoded fluorescent proteins... them to localize to the synapse and minimize extraneous fluorescence from other regions of the cell. ...synaptic maturation pattern of hippocampal adult-born granule cells over time that were not previously possible... al., 2020). Figure 3: BaF3 cells were transduced with H2B-FT virus and grown in varying concentrations...lentiviral CRISPR library that targets human glycosyltransferase genes and other proteins related to cellular...

...receiving the translation, Addgene contracted with a professional scientific translator, L. Denise Roche...101: Una herramienta de edición Addgene tiene una gran audiencia internacional, incluido trabajando con...101 eBook is now available in Spanish!  This translation was gifted to Addgene by Daniel Mejías Sánchez...Roche, to edit the translation. We are pleased to announce that CRISPR 101: Una herramienta de edición ...  Many thanks to:  Daniel Mejías Sánchez, PhD, translator  Denise Roche, PhD, MD, editor, ldeniseroche@...

...this surface”….where the complex is docked on the membrane of the endoplasmic reticulum. And graduate students...reported cleavage site on the complementary DNA strand. And this is why we love working with researchers...”. Jacob computationally added the non-template strand to the Jinek structure so that the resulting physical...protein must have in order to scan along the double-stranded DNA, recognize a PAM sequence, allow the complementary...pairing between the guide RNA and the target DNA strand, and then activate two different endonuclease active...structure captures the displaced non-target DNA strand and shows the HNH nuclease domain swung around ...juxtaposition to its cleavage site on the target DNA strand. In addition, this model also displays the several...

... mechanism to accurately repair harmful double stranded breaks, in which nucleotide sequences are exchanged...selection marker HSV-tk is used to select against random recombinants.   Design your targeting construct...selecting for cells in which the construct has randomly integrated into the genome, a negative selection...step will kill off many of the cells that have randomly integrated all or large portions of the plasmid...negative selection marker selects against improper, random recombination into a different locus. Correctly...contain the negative selection marker, but cells with random recombination may incorporate the negative selection...and animal models, their efficiency is very low, ranging from undetectable to 0.1%. In contrast, new genome...

...over for other applications, like PCR, cloning, transfection, or long-term storage. But what about those ...inverted terminal repeats. We use NEB Stable to transform and prep high quality DNA that contains these ... in a protein expression strain, we suggest retransforming the plasmid into a strain meant for DNA purification...prepping. Be sure to stay within the recommended range of the filter or you might inadvertently lower the...concentration Low copy plasmids produce fewer transcripts of their antibiotic resistance genes and can ...for high copy plasmids in standard growth strains range from 12-16 hours, but cultures with lower copy plasmids...to Quantify Your DNA Learn to Do a Bacterial Transformation Learn How to Perform a Diagnostic Restriction...

...C-to-U and A-to-I transcriptome-wide off target effects observed in whole-transcriptome RNA-sequencing, ...would convert adenine to inosine, which the translational machinery treats like guanine. This RNA editor...amount of off-target editing. In one direct transcriptome-wide comparison, REPAIRv2 reduced off-target...deaminase ADAR2DD to deaminate cytidine and used dRanCas13b (catalytically inactive Cas13) to target the ...the RESCUE system’s ability to edit endogenous transcripts finding up to 42% editing efficiency. They note...therapeutic applications, the authors tested various dRanCas13b truncations that would allow packaging the construct...

... that may have warranted their own chapters, and certainly chapters that could warrant their own books..., and he wisely makes no such attempt. He is transparent about his own biases--many of which will be shared...non-scientists, Zimmer’s observations will likely range from mildly surprising to genuinely shocking. Mainstream...he describes the now infamous tale of Rosalind Franklin and her essential-but-unlauded role in the identification...within this section. The GFP story has its own Franklin in Doug Prasher, who originally isolated the GFP...science produced by the quieter, more reserved Franklins and Prashers will find it increasingly difficult...

...mammalian cells can perform human-like post-translational modifications that bacterial and most yeast ...be produced through a single or dual-plasmid transfection, depending on whether the heavy and light chain...they’ve been separated onto two. You’ll first transfect your cells with your plasmid(s), and then wait... from dropping the tube they are contained in (Randolph et. al., 2018). At Addgene, we ensure the efficacy... Bisson L, Gervais C, Grazzini E, Durocher Y. Transient Gene Expression in Suspension HEK293-EBNA1 Cells...A. Pathak, Christopher J. Roberts, Prasad S. Sarangapani, Protein aggregation, particle formation, characterization..., https://doi.org/10.1016/j.cocis.2014.10.002. Randolph TW, Schiltz E, Sederstrom D, Steinmann D, Mozziconacci...

...species. Discovering ways to culture, propagate, transform, store, and share all of these new organisms continues...Phenotype thus making progress on the NSF “G to P grand challenge.” Creating a productive, new scientific...evolution because electric organs arose in different branches of the evolutionary tree at least 6-7 times and...mushrooms) but there are over 1,000 species. No transformation techniques are available yet. Polistes (Amy... a system using novel vesicles to efficiently transform these cells which don’t like foreign DNA at all...One can inject plasmid DNA in the eggs to get transgenic animals. They might be immortal. Manihot esculenta...ReMOT control system (Receptor-Mediated Ovary Transduction of Cargo)  delivers Cas9 RNP to the arthropod...

...specialized strains are available to confer greater transcriptional control, assist with proper protein folding...harboring this mutation are deficient in outer membrane protease VII, which reduces proteolysis of the...Lacks functional RNaseE which results in longer transcript half-life F- ompT lon hsdSB (rB- mB-) gal dcm...Chloramphenicol (pRARE) Good for “universal” translation; contains 7 additional tRNAs for rare codons ...pLemo (CamR) Expression of toxic, insoluble, or membrane proteins  T7 Express   IPTG-inducible expression...addition of IPTG. T7 RNAP is then available to transcribe the gene of interest from a T7 promoter on the... the expression plasmid in order to repress transcription from a hybrid promoter. Although the DE3/T7 ...

...the non-profit sector are transferrable to industry such as technology transfer, communications, medical... minimum (8 U.S. Code § 1184 - Admission of nonimmigrants). The foreign worker must possess at least a...and therefore the academic H-1B visa cannot be transferred to an industry position. If you cannot get an... facility management, collaboration management, grant administration, clinical operations, and so on. ...include evidence such as proof of US government grants that the researcher is working under in the lab...new accomplishments to add. Obtain copies and translations of documents that are not in English. Create...    Additional Resources on the Addgene Blog Transferable Skills Guide 6 Steps to Submitting a Resume ...

...homology-directed repair (HDR) of a CRISPR/Cas9 induced double-stranded break. This site-directed genome editing requires...methylation-dependent endonuclease (i.e. DpnI), and bacteria are transformed with the nuclease-resistant nicked plasmid (the...products of the PCR reaction are pieces of double stranded, circular DNA with staggered nicks, recent data...final PCR products are pieces of linear, double stranded DNA with homologous ends. These homologous ends...the adenosine of the GATC recognition site. Transformation: After the PCR reaction, no ligation is required...required since the E. coli you transform your PCR products into will efficiently patch up the DNA. The ...parental plasmid provides a mean for selecting for transformants which have taken up the mutagenized plasmid....

...therapeutics - one using dCas9-SAM to activate HIV-1 transcription and destroy infected cells, the other using ...histone deacetylase (HDAC) inhibitors to increase transcription of the integrated HIV-1 genome. For a targeted...dCas9-VP64 fusion commonly used to activate transcription, but with two additional activation domains ...domains (MS2 and p65) to enhance transcriptional activation. Zhang et al. designed multiple gRNAs to target ...functional Cas9 nuclease, so there’s no potential for aberrant DNA cleavage. Direct HIV-1 cleavage allows T-cells...rates of off-target cleavage. In both cases, translating the in vitro work into an animal model comes ...cleavage. Despite the potential difficulties in translating these findings into a therapy, these papers present...

...working full-time, and have received a research grant in the last five years as polled in the American...climate change page on whitehouse.gov, freezing grants, and proposing to defund aspects of research at...following subjects: Climate change Vaccines GMOs Transgender issues Reproductive health Immigration Pollution...while many also seek to honor and credit women, immigrants, and minorities who have had great impacts on...encouraged by “Broader Impacts” requirements for certain grants) (28). As one young scientist, Matthew Niederhuber...Post: Trump administration tells EPA to freeze all grants, contracts The Guardian: Trump to scrap Nasa climate... Scientists Find a Voice at Massive Rally for Immigrants National Science Foundation: Universities Report...

...of different fluorescent protein are available (Cranfill et al., 2016 and FPbase.org). A number of variants... organelles (Figure 1), showing that its acid tolerance is maintained in cells.   GFP is big GFP is a ...specific RNA stem loop-structure (Querido and Chartrand, 2008). Finally, a nuclease-deficient Cas9 can...clearly the case when proteins undergo post-translational modification with fatty acyl chains. For instance...Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells." Science 296.5569 ...28360214. PubMed Central PMCID: PMC5385933. 6. Cranfill, Paula J., et al. "Quantitative assessment of ...PMC2644460. 12. Querido, Emmanuelle, and Pascal Chartrand. "Using fluorescent proteins to study mRNA trafficking...