...collection of pathogenic Klebsiella and Pseudomonas isolates causing urinary tract infections. This project... for plant compounds to treat complications of diabetes Gloria Otito Izu, PhD Candidate, University of... the major pathophysiological complications of diabetes mellitus. Recombinant human proteins that play...play a role in insulin regulation and diabetes will be expressed from plasmids (find more information on...of the plant compounds on these proteins will be tested. Investigating changes in neuronal activity after...Armenia Gohar Tsakanova is a Senior Researcher who investigates the molecular and cellular etiopathomechanisms...projects progress. Subscribe to the Addgene blog for updates and future collaborations. Many thanks to all of...

... an entire experiment and delegate tasks to lab mates or to discuss a potential new research direction... regular check-ins with their employees. 15-30 minutes weekly or monthly will usually suffice to keep ...Managers will get a sense of the employee’s workload, updates on current projects, and offer feedback. The employee...anything that is missing.  During the meeting, take notes on the agenda points. At Addgene, we like to do ...Before leaving the meeting, skim back through your notes and pick out the action items. Move them into a ... positive peer pressure to make sure everyone completes their assigned items to keep the project moving...scope” above) or if you can cut it down to thirty minutes. Keep it moving: If you tend to get stuck diving...

...a versatile tool developed for use in prokaryotes and eukaryotes, works via direct activation. In this...conformational change that removes it from lacO sites within the promoter and ceases repression of the... absent, regulatory protein AraC binds O and I1 sites upstream of pBad, blocking transcription. The addition...addition of arabinose causes AraC to bind I1 and I2 sites, allowing transcription to begin. In addition to...CAP) can also stimulate AraC binding to I1 and I2 sites. Supplementing cell growth media with glucose decreases...sensing protein YFI. When light is absent, YFI phosphorylates FixJ, which binds to the FixK2 promoter to ...

...processing, i.e. tRNAs or Csy4 cleavage sites. This eliminates bulk from crRNA expression plasmids resulting...et al   Multiplexed Lentiviral Expression Cassettes lentiviral transduction One vector expresses... own promoter   Kabadi et al Csy4-Cleavable Cassettes transfection gRNAs expressed from a polycistronic...co-transfection of Cas9 Nissim et alTsai et al PTG Cassettes transfection gRNAs expressed from a polycistronic...s ability to process its own pre-crRNA arrays eliminates the need to include multiple promoters to drive...Multiplex gene editing with CRISPR-Cpf1 is one of the latest developments in CRISPR technologies.  It’s a simple...

...What is the universal thing that unites all scientists? Curiosity? Passion? A love of learning? No. Or... Or at least not entirely. What truly unites us all is the frustration of an experiment failing because...experiment (labeling tubes never takes longer than 5 minutes, right?) As a result, I’d always end up stressed...differently.  Tools to Try  Checklists Found in surgical suites, air fields, and military bases, checklists are... at as you work. Draw your plate maps onto your plates, alongside your physical copy. If you’ve got messy...workspace and your workflow can have a huge impact. Test different approaches, like whether it's better for... forever grateful, I started setting aside 3-5 minutes to sit quietly at my bench before the start of ...

...of animal testing and modifications. The premise of the novel involves Dr. Moreau who creates human-animal...acid recognition sites had to be designed each time we wanted to target new genomic sites. The discovery...proteins are characterized by large DNA recognition sites, which makes them highly specific.  Jurassic Park..., CBE and ABE have obviated the indel formation rates that are typically associated with the DSBs and ...that were “affected by CRISPR.”  Prime editing The latest breakthrough in genome editing is prime editing...intracellular pangenesis to the rediscovery of Mendel. Comptes Rendus de l’Académie des Sciences - Series III ...

...DNA-editing software which integrates our database of over 2000 primers. Two 96-well PCR plates are prepared with...samples through our lab using barcoded tubes and plates. The critical pieces of information for a given...functional mutations, antibiotic resistance, restriction sites used during cloning, and most importantly, the predicted... in-frame protein fusions, and multiple cloning sites. In the simplest case we have a standard backbone... purifying the DNA and gives comparable success rates and sequence quality, which is critical for the ...100 µl of glycerol stock to each of 2 96-well microplates (identical copies, see image above). We then ...

...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...

...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...

...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...

...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...

...species and those that can shuttle between prokaryotes and eukaryotes. Conclusion I still think it’s pretty...can incorporate foreign DNA into plant genomes. Eukaryotes are a bit more complicated. Yeast vectors may... resistance systems don’t always translate to eukaryotes. For example, beta-lactam antibiotics like penicillin...penicillin and ampicillin inhibit cell wall synthesis. Eukaryotes, with no cell walls, can grow alongside these...important cellular pathways shared by bacteria and eukaryotes. For example, puromycin inhibits protein synthesis...can protect both plasmid-carrying bacteria and eukaryotes. But be careful — antibiotic mechanisms vary!...

...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...

...search-and-replace” genome editing technique that mediates targeted insertions, deletions, and all possible...without double-strand breaks (DSBs) or donor DNA templates.  Figure 1: An overview of prime editing...editing, as it can edit near to or far from PAM sites, making it less constrained by PAM availability ...over 30 base pairs for prime editing. Since PAM sites occur on average every ~8 base pairs on either DNA... up to 45-fold) higher across different genomic sites compared to PE1 (Anzalone et al., 2019). PE3: Resolving... DNA to favor the edit Once the prime editor incorporates the edit into one strand, there’s a mismatch...additive improvements. These variants are worth testing if you want to make your prime editing as efficient...

...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...

...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...

...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 ...