...realized I wanted a career in science outreach: engaging the public to promote an awareness and understanding...to use to promote future attempts and learned a bit about recruiting scientists to outreach.  Try new ...spray paint to make a trophy for the winner. We promoted the contest for about two months, and we got one...network and graduate school resources daily to promote CINSO events and recruit speakers and participants... executive director of Central Indiana Science Outreach and a Postdoc at Indiana University School of ...founding a nonprofit called Central Indiana Science Outreach, or CINSO. We organize fun science events for ...supportive mentor and graduate office. I cannot stress how important they were to my success as a student...

...Effective science communication is required to promote public support for research and to keep useful ... a graduate student organization that works to promote science communication. Science in the News hosts...and SITN The Most Difficult thing Vini, Amy, and Outreach Scientist Jessica Welch Have Had to Explain Importance...

...plasmids, the MCS is often downstream from a promoter. Insert Gene, promoter or other DNA fragment cloned...cloned into the MCS for further study. Promoter Region Drives transcription of the target gene. Vital...

... a tool that can help you advance your career, promote career growth, and/or switch your field of work...is larger and there are more opportunities for promotion. It’s not uncommon for bachelor’s and master’s..., i.e. Principal Scientist or Scientist I/II. Promotions don’t happen overnight though, and it might take...but in time I realized its value. My master’s strengthened my critical thinking and problem solving abilities...

...design your donor template to include a T7 promoter upstream of the left homology arm, perform in vitro...transcription to create your ssDNA. Note that the T7 promoter will not be included in the final ssDNA template...GG); this design has been shown to increase T7 promoter activity. To avoid re-cutting by Cas9 after editing...containing the homology arms on both sides and the T7 promoter on one end. Typically, Easi-CRISPR works best ...Addgene plasmids. Design PCR primers to add T7 promoter-left homology arm and right homology arm to the...template with 55-100 base homology arms upstream and downstream of the insertion site. If you are using...

... may differ (e.g., a set of various mammalian promoters), the same cloning procedure can be used to incorporate... in this method include fluorescent proteins, promoters, enhancers, polyadenylation signals, inducible...Table 1). Each construct was flanked with a CMV promoter (to drive high-level expression) and a polyA signal...multiple identical polyadenylation signals or promoters Gibson et al., 2009 MXS-Chaining, BioBricks,...and different methods are suited for particular downstream applications. The tradeoffs between various cloning...

...induce transcription at CpG regions of various promoters. Other researchers additionally fused a TAL effector...: dCas9-p300 adds H3K27ac marks to histones in promoter and enhancer regions. These marks are associated...Figure 2: dCas9-Tet1 demethylates cytosines at promoters and enhancers. This targeted demethylation is ...Figure 3: dCas9-DNMT3A methylates cytosines at promoters and enhancers. This targeted methylation is associated...-based acetyltransferase activates genes from promoters and enhancers. Nature Biotechnology 33:510–517...in the DNA oxidation and repair pathways work downstream of Tet1 to restore the DNA after cytosine removal...

...optimism they encapsulate, and the solutions they promote help motivate you to spend a little time advocating...fight is far from over here at home.  The work of promoting and defending the role of science in our democracies... take in abusing climate science skepticism to promote harmful policies that benefit polluting industries.In...marching in and of itself will be enough. IT WON’T! Promoting the incorporation of science in policy, demanding... that uses yeast cells to study how DNA damage promotes cancer. She is also a science communicator, UBC...Hundreds of scientists and supporters in Montreal took to the streets on April 22 to join in the March for...at the Goodman Cancer Research Centre at McGill University in Montreal, Canada.  Outside the lab, she ...

...Regulated Cre expression – placing Cre downstream of promoters that are active only in certain cell or...examined a number of important plasmid elements – promoters, origins of replication, protein tags, and antibiotic...Cre under a variety of ubiquitous and regulated promoters, and many loxP-containing alleles have also been...often called a “lox-stop-lox” or “LSL” cassette) upstream of a gene of interest will prevent gene expression...

...repeat (LTR) of the HIV-1 genome, which acts as a promoter, and found that SAM targeting of the enhancer ...NF-KB binding sites increased activation of HIV promoter-luciferase constructs. In multiple HIV-1 latent...cells with activated HIV-1, measured via HIV-1 promoter-driven GFP expression. In cell lines that can ...indels may not ablate viral function, but rather promote resistance to CRISPR/Cas9. While these results ... acquired immunodeficiency syndrome (AIDS) in untreated individuals. Symptoms of AIDS include rapid weight... viral levels will soon skyrocket back to pre-treatment levels due to the viral reservoirs located in ...in latently infected cells. Although ART-treated patients lack plasma HIV-1, they are at increased risk...

...-inducible promoter, though Khetrapal et al. also demonstrated that the arabinose promoter worked too.... under both rhamnose- and arabinose-inducible promoters. Tight control of toxin expression is absolutely...a rhamnose-based tightly regulated and tunable promoter system." Biotechniques 40.3 (2006): 355-364. PubMed...routinely clone the purified amplicons upstream and downstream of the desired deletion directly into cut...transcriptional terminator BBa_B1002 (from IGEM) upstream of the MCS to reduce transcriptional readthrough...accessibility, selective RNA unfolding and sliding at upstream standby sites." Nucleic acids research42.4 (2013...

...resources and information about organizations promoting reproducibility in this booklet (which was also...line we may see further adoption of policies that promote reproducible publishing practices. One example ... also hopefully see a shift toward hiring and promotion practices that focus on reproducibility and ensuring... Including descriptions of many organizations promoting reproducibility List of Tools for Reproducibility...academia, industry, nonprofits, and publishing. The livestream video from the event can be found below along...interested in, but I’d recommend watching the whole livestream for a more holistic understand of reproducibility...reasons we alter these designs if we do so. The most extreme version of this (and possibly the most effective...

... site accepts the sgRNA target sequence for U6 promoter expression and the second site accepts the homology... a direct fusion of the desired gRNA to the U6 promoter, and a second plasmid containing the homology ...regions of homology to the gene of interest to promote homology directed repair after a CRISPR/Cas9 induced...T2A-TurboGFP-PEST), and small epitope tags (HA, Myc, Strep tag II, AviTag, HaloTag, SpyTag). The Foerstemann... N-terminal tagging vectors (eg GFP, Flag, YFP, Strep, TEV-V5) with either blasticidin or puromycin selection...

...fluorescent protein eYFP from the hSyn1 promoter Viviana Gradinaru 117383-AAV1 TRE-DIO-eYFP An AAV genome with tet-inducible...-mCherry Expression of hM4D(Gi) using the CD68 promoter. Bryan Roth 75033-AAV9 pAAV CD68-hM4D(Gi)-mCherry...mCherry Expression of hM4D(Gi) using the CD68 promoter. Bryan Roth 83894-AAV1 pAAV-hDlx-Flex-dTomato-Fishell...An AAV genome that expresses tTA from the hSyn1 promoter that contains a positive feedback loop for amplifed...Cre-dependent expression of the tTA from the hSyn1 promoter. Contains a positive feedback loop for amplifed...recombinase-dependent expression of mNeonGreen from the CAG promoter Viviana Gradinaru 100798-AAV1 pAAV-syn-FLEX-splitTVA-EGFP-tTA...expression of the fluorescent protein EYFP from the CAG promoter Viviana Gradinaru 104055-AAV2 pAAV-CAG-eYFP An...

.... Pro-Tip Different brands and lots of FBS can promote or inhibit transfection. Test a variety of brands...control the multiplicity of infection (MOI) in downstream studies. This protocol was developed for A549...Microcentrifuge tubes, Neptune 3745.X 6-well tissue culture treated dish, Corning 3516 15 mL conical tubes, VWR 21008... after 20–30 passages. Do not add penicillin/streptomycin to the media, as it can affect viability and...target cell line needs to be empirically determined. Treat the target cells with a range of doses of antibiotic...vector pRosetta . The following day, cells were treated with puromycin. Colonies were allowed to grow in...EF1α dCas9-KRAB . The following day, cells were treated with puromycin. Colonies were allowed to grow in...

...experiment is determined by the capsid and promoter/enhancer strengths in the cell type of interest as well ...transgene expression. Gene regulatory elements [promoters and enhancers, miRNA target sites, and recombination...over systemic delivery. Otherwise, cell-specific promoters and enhancers (Mich et al., 2021) can help restrict...optogenetic and chemogenetic tools. Dual expression Promoters, miRNAs, enhancers, and switches all take up precious...candidate regulatory elements such as enhancers and promoters for cell type-specific targeting (Chan et al.,...when paired with appropriate cell type-specific promoters or Cre lines. Spatial transcriptomics suggests...AAV-PHP.eB, when paired with the ubiquitous CAG promoter, is relatively biased toward L5 and inhibitory...

... repression of transcription through binding a promoter region to inhibit association of transcriptional...targeting their genomic locus of interest, the promoter region of the transcription factor IRF-1. Immunoprecipitation...lab found a microRNA (miR483) that binds to the promoter of the growth factor IGF2 open reading frame to...dCas9, with bound DNA, can then be purified by streptavidin high-affinity purification. This system is both...

...are therefore good temporal reporters to measure promoter activity, for example. Useful positive controls...dark after the transformation. Use introns Plant promoters can be recognized or leaky in Agrobacterium. This...of the GUS gene with the commonly used CaMV 35S promoter in Agrobacterium. This can be easily avoided by...more easily quantify the effect of the different treatments on transformation success. Some reporter gene...

...However, the same technique can be used to move promoters, selectable markers, or any other DNA element ...single enzyme, but this will require phosphatase treatment of your recipient plasmid as well as a specifically...re-circularization of the recipient plasmid. You should treat your digested recipient plasmid with a phosphatase...

... researchers must be extreme self-starters and especially good at self-promoting—No one is going to tell...diversity and acceptance can be an issue in getting promoted (especially race and gender) Interpersonal issues...especially with peer mentors, to discuss your strengths, weaknesses, interests and passions is another...

...limited by the lack of well characterized inducible promoters. Far removed from the bio-industry’s vision of... the same dCas9 protein. By using an inducible promoter to modulate dCas9 expression, multiple scRNA transcriptional...genes or to introduce heterologous genes, but the trend of research has shifted in recent years toward tinkering...regulators (Zalatan et al. 2015).  These scaffolds are stretches of non-coding RNA interspaced with linkers that...

... °C. Pro-Tip Different brands and FBS lots can promote or inhibit transfection. Test a variety of brands...Microcentrifuge tubes, Neptune 3745.X 6-well tissue culture treated dish, Corning 3516 15 mL conical tubes, VWR 21008... after 20-30 passages. Do not add penicillin/streptomycin to the media, as it can affect viability and... reagent for your target cell line. To do this, treat target cells with a range of doses of antibiotic... try higher/lower dilutions depending on your downstream applications. If you’ve titered your virus beforehand...

... sensor, he will construct plasmids containing promoter regions that activate fluorescent protein transcription...and to Seeding Labs who helped us coordinate and promote the awards. Additional Resources on the Addgene... has studied various aspects extreme life on earth including extremophile microorganisms, biomarkers, ... to pyrazinamide (PZA) (a drug commonly used to treat tuberculosis) and in identifying new drug targets...biofilm formation. The quest for plant compounds to treat complications of diabetes Gloria Otito Izu, PhD...infusions of L. taraxacifolia leaves are widely used to treat several diseases. Her goal is to identify and isolate...blocked, resulting in reduced blood flow. Novel treatment options, as well as non-pharmacological solutions...

...activated, this DREADD acts through the Gq pathway and promotes neuronal firing. Plasmid Serotype Name Depositor...more accurate cell and tissue targeting. While promoters and AAV serotypes can sometimes be used to target...2-N-2xStrep-IRES-Puro: Expression of the SARS-CoV-2 N protein  pLVX-EF1alpha-SARS-CoV-2-M-2xStrep-IRES-Puro...pLVX-EF1alpha-SARS-CoV-2-E-2xStrep-IRES-Puro) and NSP2 (pLVX-EF1alpha-SARS-CoV-2-nsp2-2xStrep-IRES-Puro) are also...SARS-CoV-2 M protein pLVX-EF1alpha-SARS-CoV-2-orf3a-2xStrep-IRES-Puro: Expression of the SARS-CoV-2 ORF3a ...

...expression plasmids in a high copy backbone with a T7 promoter driving multiple combinations of WT, codon optimized...which drive expression using either TEF1 or GAL1 promoters. These vectors are intended for the high level...race to find vaccines, therapeutics, and other treatments to stem the spread of COVID-19. That’s why we...sites (thrombin and TEV), and C-terminal tags (StrepII, 3xFLAG, 6xHIS, MBP, Myc). These constructs include...expression and purification of SARS-CoV-2 proteins for downstream experiments and offer flexibility in tag, cleavage...requested the plasmid. By leveraging our core strengths of intensive screening and the use of high throughput...

...dCas9 along with transcriptional repressors to the promoter region of the gene of interest. dCas9-based repressors... and a dCas9-KRAB fusion, both targeted to the promoter region. Cas9 activators include Cas9 fused to ...the Suntag array of factors, all targeted to the promoter region.   Ok, I have a Cas9! What do i do ... genome.  2) The target is present immediately upstream of a Protospacer Adjacent Motif (PAM), the sequence...

...different promoters followed by leader sequences (encoding small leader peptides) downstream of which ... vectors within this kit contain a variety of promoters to facilitate gene expression in bacteria, yeast...different genes was predictable based solely on the promoters and Shine-Delgarno sequences used in this so-called...transcription from resulting in the production of a downstream peptide, the transcription terminators in this...BIOFAB Plasmid Set contain terminators of varying strengths centered within GFP/RFP bicistronic reporters ...

...targeted gene from its promoter, creating a loss-of-function allele as well as a promoter-FP fusion for the...for the CRISPR/Cas9 revolution!   References Streamlined Genome Engineering with a Self-Excising Drug ...

...unstable fluorescent proteins under the control of promoters responsive to water or nutrient levels can help...fluorescent protein reporter under the control of promoters that control cell cycle gene expression.  Additional...serotonin reuptake inhibitors are frequently used to treat depression and anxiety, but these drugs are often...

...for its powerful promoter of a gene important for methanol usage. This gene promoter, AOX1, was co-opted...Affordable to culture and easy to work with Grows extremely fast (doubling time of ~ 2 hours) Amenable to...

...that flank the gene cassette, which contains a promoter, the gene of interest, and a polyA signal. Created...sequences are from AAV2 because they demonstrate high promoter activity (Earley et al. 2020), but non-AAV2 ITRs...limited, but this area of research is important for downstream AAV applications.  Tips & FAQs Due to their high...

...efficient AAV prep for a 4.2 kB insert with the CMV promoter using the AAV-BR1 capsid? A: It is our understanding...4.7kB.  If your insert alone is 4.2 kB and CMV promoter+enhancer is 0.5 kB, then you would be oversized... reliable quantitation. Differences in sample treatment across methods can affect reported titer by either...

...search scoped to viral preps shows filters for promoter and serotype, among others. We also created a ...example, there is only one viral prep filter for promoter “Syn”, that, when selected, will match variations...members from the Customer Service, Data Curation, Outreach, Product, Scientist, and Software teams. We knew...

...protein whose coding sequence is closest to the promoter will be expressed. This system differs from Brainbow...translation-blocking STOP cassette immediately following the promoter. The STOP cassette includes a mutant YFP that .../FRT recombinase. When placed under different promoters, Brainbow and Flpbow can be used to label distinct...Drosophila. Why Brainbow? Brainbow highlights the trend in biology towards single-cell resolution imaging...

...suitability of various antibiotic resistance markers, promoters, and inducible gene expression systems in bee ...fusion is under control of the arabinose-inducible promoter. Cells are first induced with arabinose to begin...designed to bind and detect nearly any RNA sequence. Downstream of the hairpin is a ribosome binding site (RBS...responds to infection, dysbiosis, and antibiotic treatment. To facilitate these studies, Pamela Silver’s ...

... Atlas (NURSA), one of our primary goals is to promote an understanding of how these processes are regulated...upon a context defined by ligand, cell type, and promoter, organizing the expression of functional groups...transported from cells, traverse the body through the bloodstream, and eventually elicit their actions on other...drugs currently available or being developed to treat a multitude of therapeutic indices, including hypertension...

...either the Thy1 or CAG promoter, while Brainbow AAV is under control of the EF1a promoter. Livet et al., ...known as SpyLigase and is a protein domain that promotes the formation of an isopeptide bond between 2 ...insert fragments of DNA containing basic parts (promoters, UTRs, coding sequences, terminators, etc) into... creating a single transcriptional unit (Ex: a promoter, 5’UTR, coding region, and terminator). Next, ...individual types of pre-cloned insert modules (plant promoter, N-terminal tag, coding sequence of the gene of...Biotech. 2013. Dec 23.   Highly bright and extremely dim GFPs The availability of the three dimensional...unprecedentedly-high brightness, bfloGFPa1, and the other with extremely-dim brightness, bfloGFPc1. The plasmids used for...

...customizable sgRNA from the U6 promoter and puromycin resistance from the EF-1a promoter. It’s a 3rd generation...plasmid from the Zhang lab expresses Cas9 from Streptococcus pyogenes with an invariant sgRNA scaffold and...

...kit contains 96 parts including terminators and promoters, CRISPRi parts, selectable markers, transformation...determined protein expression levels from different promoters, and inhibited gene expression using CRISPRi. ... acceptor vectors from the MoClo Toolkit from Sylvestre Marillonnet’s lab to develop synthetic biology...

...vector containing homologous ends to the breakpoint promotes homologous recombination of the targeting vector... process. The addition of small molecules that promote homologous recombination or the use of long single-stranded...recessive traits and their impact on diseases or treatments, but are not useful for genetic engineering. ...initially inhibited by the presence of a stop cassette upstream of the target gene in the targeting vector. The...

...expresses multiple reprogramming factors driven by one promoter. While lentivirus has similar rates of reprogramming...control. Additionally, Sendai virus doesn’t rely on promoters for transcriptional regulation, so it requires... mini-plasmids. They contain only a eukaryotic promoter and the cDNA(s) to be expressed and they don’t...phosphatase, expression of embryonic proteins, and promoter demethylation and expression of endogenous pluripotency... cellular defenses against ssRNA: phosphatase treatment, substituting cytidine with 5-methylcytidine, ...

...very realistic. Envisioning GFP as a substrate promoting dimerization, Tang et al. tested pairs of their... they activate gene expression from different promoters. Moving to an in vivo system, Tang et al. electroporated...at LoxP sites. Placing a lox-STOP-lox cassette upstream of a gene of interest blocks transcription when...

...neuron-specific expression (hSyn promoter) and pan-mammalian cell expression (CAG promoter). An improved version ...distinct cell subtypes for efficient expression and downstream monitoring. This targeted approach allows scientists...

...allows for customization with strain-specific promoters, antibiotic resistance selection markers, gRNAs...nature allows for the customization of guides, promoters, selection markers, and different versions of ...trimethoprim when compared to control gRNA treated or untreated bacteria. Had the folA gene product not been..., Oren Rosenberg, and Carol Gross provides a streamlined suite of tools for using CRISPRi in many types...tri-parental mating scheme. Mobile-CRISPRi integrates downstream of the highly conserved glutamine-fructose-6-...and right Tn7 end sequences (green) integrates downstream of the glmS gene in the recipient's genome. Antibiotic...were grown in presence of trimethoprim, cultures treated with a folA gRNA were more sensitive to trimethoprim...

...plant lab, so it includes everyone’s favorite promoters, plus a few new ones that are not used so much...know how to start. They might want to use our promoters and terminators, but then they aren’t sure how...motivates her research, the MoClo Kit she and Sylvestre Marillonnet share with the scientific community...with the Athena SWAN Charter and it has really strengthened the network of female scientists. Twitter has..., Jonathan D. G. Jones, Nicola J. Patron, and Sylvestre Marillonnet. ACS Synth. Biol., Just Accepted Manuscript...

... the pYES2.1 plasmid is controlled by the GAL1 promoter. The cloning procedure adds both V5 and His6 epitope...grow in the absence of methionine. Since the GAL1 promoter controls MET/Met gene expression, complementation...Associate Professor at Boston College. National reports stress the importance of providing authentic research ...

...Addgene are perfect for expressing GFP from a given promoter, and the technique can be adapted to other transgenes...observed will be dependent on both the endogenous promoter and the 3’ UTR of the locus of interest, since...of a GFP-Puro cassette into a given locus, just upstream of a stop codon. Briefly, the PITCh vector should...since the GFP-Puro will be inserted just upstream of a stop codon. One potential concern is if the GFP-Puro...

... VchINT, three separate plasmids and multiple promoters were used to express the necessary components ...required genes and the CRISPR RNA under the same promoter, and a donor plasmid (pDonor) encodes the mini-transposon...labs (Peters et al., 2017; Klompe et al., 2019; Strecker et al., 2019), which direct RNA-guided transposition...Halpin-Healy et al., 2020). Genomic targets require an upstream 2-bp PAM sequence for recognition by QCascade;...from the donor molecule, and inserts it ~50 bp downstream of the genomic target site bound by QCascade ...structures known as cointegrates (Vo et al., 2020; Strecker et al., 2020).  CRISPR-transposons for bacterial...et al., 2019). Recently, the lab introduced a streamlined expression approach (Fig. 2A), involving two-...

... and triggering release of the stress hormone norepinephrine to promote vigilance. A recent study investigated... "Dorsal raphe neuroinflammation promotes dramatic behavioral stress dysregulation." Journal of Neuroscience...readout for associated emotional phenomena like stress levels, but researchers have to be mindful of motivation...coeruleus (LC) is known to play a major role in the stress response circuit by integrating neuronal signalling...freezing response, a form of immobility caused by stress and fear. Such a behavior can be monitored in an... 1970s to test the efficacy of antidepressant treatment in rodents. In this test, an animal is placed ... cortical noradrenergic excitability upon acute stress." The EMBO journal 37.21 (2018). PubMed PMID: 30209240...

...10878/ . Description Vector Element U6 Human U6 promoter drives RNA Polymerase III transcription for generation...transduced cells. hPGK Human phosphoglycerate kinase promoter drives expression of puromycin. Puro R Puromycin...with effective gene silencing: Starting at 25nt downstream of the start codon (ATG), search for 21nt sequences...position 15-19. Avoid targeting introns. Avoid stretches of 4 or more nucleotide repeats, especially repeated...Bovine Serum (FBS) Invitrogen: #16000 Penicillin/Streptomycin Invitrogen: #15140-122 Polypropylene tubes VWR...be passaged in DMEM + 10% FBS with penicillin/streptomycin, cells should be plated at this step in DMEM...10% FBS without antibiotics (no penicillin or streptomycin). Day 2: b. Perform the transfection in the ...