...functional units which are transcribed together. Image courtesy of Robert Hurt. Understanding how individual...Figure 2: The three assembly levels of MoClo. Image courtesy of Rob Hurt. What makes MoClo systems so powerful...optimizing a TU is much easier when you can quickly test that part in different contexts and plug different...products. The flexible nature of MoClo allows users to test a variety of different orientations and components...best promoter for a given peptide can easily be tested by generating Level 1 TUs with unique promoters...modifying enzyme) to pair with it can be queried by testing any number in the Level 2 assembly. These pathways...attractive! It allows researchers to assemble and test many variables all at once. CRISPR CRISPR arrays...

...The job of scientists to observe how things work, test theories, and solve problems is supposed to be impartial...ultimately galvanized scientists to rally in Ottawa to protest the “death of evidence,” and to speak out in unprecedented...Nashville, TN, USA). I had never participated in a protest before, so I had no idea what to expect. Admittedly...although there was at least one person vocally protesting us, he was not threatening in any way. Participating...studies beta-cell function and failure in Type 2 Diabetes. Birmingham, AL, USA 33.5207°N 86.8025°W - Jonathan...March, three climate scientists and an economist testified before the House Science Committee about climate...first step, and continue this work by voting for candidates that respect science, learning to communicate...

...several labs were able to decipher the journey of lymphocytes from the bone marrow to the thymus where they... tagged with a fluorescent protein or dye. It separates the cells mechanically in a vibrating nozzle, ...How do T cells and/or Neutrophils travel to the sites of infection or damage (Peters et al. 2008, Chtanova.... 2008, Kamenyeva et al. 2015)? How do blood monocytes patrol blood vessels during immune responses (...Salomonnson et al. 2012). Finally, it is worthwhile to test the photostability of your fluorescent protein when...flank fluorescent proteins in a plasmid with loxP sites such that their expression is turned on or off in... blood vessels and tissues by a population of monocytes with patrolling behavior." Science 317.5838 (2007...

...restriction enzymes. These cut outside their restriction sites allowing you to easily create customized overhangs...pooled in one tube and ligated in as little as 30 minutes! Alternatively, you might consider Gibson cloning... vector and your insert, DpnI digest away the templates, and transform the PCR products into E. coli. ...cut and ligation processes happen in a matter of minutes. Cost: Funding is usually limited - what cloning...will require a PCR reaction or two to generate templates for the cloning process. Time spent actually doing..., because they will come with comprehensive and tested protocols, as well as premixed and ready-to-use...construct does not contain the type IIS recognition sites, allowing you to keep the type IIS enzymes in the...

...liquid media or solid media (on plates). In general, amplification on plates can help to ensure that diversity...cheaper and easier. One library that Addgene distributes with a chemical transformation amplification ...normal transformation protocol, the size of the cuvettes your lab’s electroporator uses, etc. The DNA amount... anywhere from 10 ng to 1 μg of DNA. It often correlates with the size of the library, but generally needs...library varies significantly in plasmid size). On plates, each transformed bacteria will be its own colony... different, and you should always experimentally test the protocol you design to ensure that it works ...

...This post was contributed by guest blogger Tessa Cressey. The highly pathogenic Ebola virus belongs to... into the infected cells. The EBOV polymerase replicates the viral genome and transcribes viral genes.... is encapsidated by the nucleoprotein, NP. NP associates with the viral polymerase, which is formed by...mRNAs are translated) by cellular machinery. NP encapsidates the negative sense, single-stranded RNA minigenome...replicate the minigenome, the viral polymerase initiates RNA synthesis at the 3’ end of the minigenome ...polymerase recognizes the gene start signal and initiates transcription of the reporter gene. The polymerase...that various viral components play in disease. Tessa Cressey is a PhD Student in the labs of Dr. Elke...

...search-and-replace” genome editing technique that mediates targeted insertions, deletions, and all possible...without double strand breaks (DSBs) or donor DNA templates. How does this work? First, an engineered prime... up to 45-fold) higher across different genomic sites compared to PE1. The pegRNA: A template and guide... DNA to favor the edit Once the prime editor incorporates the edit into one strand, there’s a mismatch...genome editing as it can edit near or far from PAM sites making it less constrained by PAM availability like...over 30 base pairs for prime editing. Since PAM sites occur every ~8 base pairs on either DNA strand, ...efficiency in HEK293T cells with 1-10% indels. In other tested cell types, including post-mitotic primary mouse...

...growth rate conditions, E. coli preferentially upregulates production of tRNAs that recognize codons found...regions of the protein may be translated at different rates. The ribosome will move quickly along regions calling...be many possible ways to encode them and what constitutes the “appropriate” codon is not necessarily obvious...tRNA abundance Repetitive sequences Restriction sites Sequences prone to create secondary structures in...choice may interrupt transcription factor binding sites) As you might imagine, it’s not easy for humans... due to problems such as mismatched translation rates and potential effects on cell growth, even hosts...23262490. PubMed Central PMCID: PMC3565066. 7. Quax, Tessa EF, et al. "Codon bias as a means to fine-tune gene...

...interaction for all of us studying human genes in test tubes and gels). As a freshman in college I studied...beneficial for many systems. How the EDGE Program promotes scientific sharing The EDGE Program is designed...approaches, and infrastructure necessary for direct tests of cause and effect hypotheses between gene function...Caterpillars that eat milkweed generate cardenolides that tastes yucky (and is poisonous) for bird predators - birds...for the study of basic evolution of fungi and eukaryotes and includes the fungus that is killing frogs...No transformation techniques are available yet. Polistes (Amy Toth) These wasps are like the ones that ...actually rare in the animal kingdom. Gasterosteus aculeates (Dan Bolnick) A paper on stickleback fish science...

...of most CRISPR systems and generates double strange breaks at target sites 5' of the 5'NGG3' PAM sequence... version of CAG promoter that the Zhang lab has tested in a variety of mammalian cell lines. Cas9, of ... for more information). V2.0 This designation separates pSpCas9(BB)-2A-Puro (PX459) V2.0 from it's predecessor... has a U6 promoter followed by BbsI restriction sites into which you can clone your 20 nucleotide guide...FLAG tag at its N-terminus. This tag can be used to test for SpCas9 expression post transfection by western...

...start defining keywords you’ll want to find in candidates’ resumes or CVs; “vivarium”, “weaning”, “colony...will help save time by screening out irrelevant candidates, and, more importantly, it’ll help you define...personnel salary ranges, go to online job information sites (like, indeed.com, or salary.com). Search industry...is important to ensure that you’re looking at institutes that have the means to support your work. For...work requires specialized equipment, try finding institutes that offer shared equipment that you can use ...the anxiety that comes with negotiations. This creates a self-assured discussion when it comes time to...faculty position because none of the offering institutes were able to provide the resources required for...

...advantages over antibodies (Ahmed et al., 2012, Bates and Power., 2019): rapid blood clearance, which ...likelihood to aggregate due to their small size (Bates and Power., 2019). Their rapid clearance from blood...therapies in clinical trials or already in the clinic (Bates and Power., 2019).Many of the scFvs designed for...intracellular T cell receptor portion of the CAR facilitates signal transduction and release of cytotoxic ...2012:1–15 . https://doi.org/10.1155/2012/980250  Bates A, Power CA (2019) David vs. Goliath: The Structure... (1988) Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin...research from the Trimmer lab Check out Addgene's latest project to develop an open antibody resource ...

...biotin-binding sites on this complex can bind to your biotinylated antibody. This creates a large meshwork...absorb most of the biotin they need in the small intestine from the microflora that reside there. Biotin ... organic acids to produce important cellular metabolites. Biotin acts as a carboxyl carrier from one compound...observed that animals fed a diet of primarily egg whites would develop skin lesions and other terrible health...for easy detection. The multiple biotin binding sites found on avidin are also ideal for signal amplification...of the E.coli BPL, BirA. If you recall, BirA biotinylates only one lysine in the entire E.coli proteome...

...closed states (A) and (B) but with much lower affinity than their open states (C). Figure courtesy of IPI...conformational states: bent-closed, extended-closed, and extended-open. In the two closed states, integrins...bind RGD-binding integrins at non-ligand-binding sites and do not interfere with integrin function. Overall...writer at the Institute for Protein Innovation. She writes about all things IPI, from the scientists and scientific...

...for seeking knowledge. The process begins with a testable hypothesis - an educated guess about how some ...of the world functions. Experiments come next, testing the correctness of the hypothesis. The results ...march in Washington, D.C., the seat of the United States government? Politics and science are intrinsically...Environmental Protection Agency, and the National Institutes of Health endanger scientists’ ability to uphold...these risks include: 1) Lost trust Perhaps the greatest risk is that the public will lose trust in scientists...scientist, Matthew Niederhuber, said, “if the march motivates more scientists to reach out to their communities...Center: The Politics of Climate Change in the United States CNBC: Kellyanne Conway: “I”m not in the job of ...

...the blocking-only templates will compete with the blocking/intended mutation templates resulting in cells...Ludwig-Maximilians-University in Munich and Marc Tessier-Lavigne’s lab at the Rockefeller University in ...have focused on strategies to improve overall HDR rates, we have shown recently that desired genome-editing...unwanted indels. Following this observation, we tested a strategy that was discussed in the field at the...described project while he was a Postdoc in Marc Tessier-Lavigne’s lab at the Rockefeller University in ... NYC.   Dylan Kwart is a graduate student Marc Tessier-Lavigne’s lab at the Rockefeller University. He...

...of Brainbow, Brainbow 3.0 (Cai et al., 2013), incorporates the highly photostable fluorophores mOrange2...Tissue-specific Cre recombination in this mouse generates cells that randomly express either nuclear GFP... to describe the self-renewal of cells in the intestinal crypt, and since then has become a powerful tool... lab also developed a version of Tetbow that incorporates chemical tags instead of XFP to optimize it ...expansion and dedifferentiation of mature pigmented melanocytes." Cell stem cell 21.5 (2017): 679-693. PubMed...PMCID: PMC6245733. Snippert, Hugo J., et al. "Intestinal crypt homeostasis results from neutral competition...PMCID: PMC4077948.  Yan, Kelley S., et al. "The intestinal stem cell markers Bmi1 and Lgr5 identify two ...

...analyze sequence tool: Annotates features, BLASTs, aligns, digests, and translates a sequence.   NEBCutter2...restriction enzyme cut sites Webcutter 2.0: For finding restriction enzyme cut sites Open reading frame finder...sequences in a database ExPASy translation tool: Translates a DNA sequence using all six open reading frames...primer design tools, but here are a few stand-alone sites for general primer design, as well as primer design...

... viruses. Now focusing on plant biology, she coordinates the Network for the Genetic Improvement of Cowpea...agricultural plant. Her current research focuses on endophytes, bacteria, and fungi that reside within living...proteins and enzymes. These compounds will later get tested for their potential agricultural and medical use...detect glyphosate via H2O2, one of the reaction’s metabolites. One useful genetically encoded H2O2 probe is...projects progress. Subscribe to the Addgene blog for updates and future collaborations. Many thanks to all of...References 1. Martinez-Klimova, Elena et al. “Endophytes as sources of antibiotic” Biochemical Pharmacology...

...separate resistance cassettes and your gene of interest is cloned into/inactivates or completely removes...translocation Bacteriostatic 5-25 ug/mL in EtOH *In prokaryotes.     **Dissolve in dH2O and sterile filter unless... are trying to disrupt or delete, which both inactivates the gene and acts as a marker for the mutation...months. Ampicillin breaks down especially fast and plates should be used witin 1 month for optimal efficiency... them to media hotter than about 55oC and store plates/stocks wrapped in foil if a light-sensitive antibiotic...