...to always look closely at the trace file (or chromatogram) of the sequencing results you get back from...facility. When it comes to DNA sequencing the chromatogram is your visual control. And, like all controls...controls, missing out is a big mistake. Chromatograms come to the rescue for analyzing and troubleshooting...But look closer, the overlapping peaks in the chromatogram suggest the results are not as certain as the... of the following programs to view your .ab1 chromatogram file 4Peaks (Mac) SnapGene Viewer (Mac/PC) ...peak, don’t be shy. Feel free to edit it. Most chromatogram viewing programs (even the free ones) allow ...allowing us to share it with our readers! The chromatograms in this article were created with SnapGene. ...

...mDlx-GFP, hDlx-FLEX-GFP, hDlx-Flex-dTomato, mDlx-GCaMP6f, hDlx-GiDREADD-dTomato and mDlx-ChR2-mCherry. Find... a cell. The Goedhart lab, Gadella lab, and collaborators have an ongoing effort to improve genetically...by one-step immobilized metal ion adsorption chromatography (IMAC) using Ni-NTA magnetic beads. Workflow...multiplexed gRNAs and of dCas9 fusions. The dCas-KRAB activator and the dCas-VPR repressor are introduced into...an EGFP reporter. Expression of the dCas9-KRAB activator or dCas9-VPR repressor in hPSCs is also driven...expression. This includes a CRISPRi-based synthetic oscillator, bistable network, stripe pattern-forming incoherent...calcium sensors (jYCaMP1), variants of the calcium indicator jGCaMP7, that are optimized for excitation wavelengths...

...controlled by still other regulatory factors: enhancers, boundary elements, insulators, and silencers; however...tetracycline operators. Transcription can be turned on or off depending on what tet transactivator is used....the promoter (RNA polymerase binding site) and operators (response elements). Promoters are about 100 to...expression Promoter from T7 bacteriophage plus lac operators Negligible basal expression when not induced. ...RNA polymerase, which is also controlled by lac operator. Can be induced by IPTG. Commonly found in pET...pET vectors. Very tightly regulated by the lac operators. Good for modulating gene expression through varied...

... introduced, stochasticity will result in a combinatorial expression of these three genes with different...inherently low. This is because the stochastic and combinatorial expression of fluorescent proteins is only possible...original method, we used the Tetracycline trans-activator system to boost the expression levels, and we ...we named the system Tetbow (Tetracycline trans-activator Brainbow) (Sakaguchi et al., 2018) (Figure 1)....sensory systems focusing on the functional and anatomical maturation of the olfactory system.      Takeshi...University. His lab has been studying functional and anatomical aspects of sensory circuit development using ...Livet, Jean, et al. "Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous...

...fully-equipped laboratory for citizen scientists. In addition to maintaining a laboratory to support both... of independent Do-it-Yourself (DIY) biology laboratories working towards enabling everyone to participate... set on top of filing cabinets, a rickety old incubator, a few pipettes, and some home-made equipment....Anyone can become a member of BosLab and previous laboratory experience is not required. Our members are skilled...have non-traditional scientists working in our laboratory! Don’t worry though, we make sure that everyone...a co-organizer at BosLab, a community biotech laboratory in Somerville MA. Follow her on twitter at @ZebraElement...

...include the laboratory personnel, the serum, the cell culture media, water baths, incubators, etc. Contamination... in a single incubator since it increases the risk of cross-contamination. CO2 incubators should be cleaned...Contamination! Tissue culture media often have indicators included in the media that turns yellow from ...proliferating shortly. Once they are present in the laboratory environment, they are difficult to eradicate....tighten the flask cap before placing them in the incubators. This will also solve the problem of contamination...airflow and the lab facility periodically. The CO2 incubators, used for maintaining cell lines, must not be...

...voltage indicator Article contributed by Shreya Vedantam Genetically encoded voltage indicators (GEVIs)...fluorescent optics. Existing red fluorescent voltage indicators, however, fall short compared to GFP-based sensors... sensors and blue-light-activated optogenetic actuators.  To overcome this overlap, Yiyang Gong’s lab ...linker between them. The Ace-mScarlet voltage indicator works like many FRET-opsin GEVIs. With an increase...channelrhodopsins. Find the Ace-mScarlet voltage indicator plasmids at Addgene! Beck et al., Sci Report 2019...cyanobacteria. This kit contains 96 parts including terminators and promoters, CRISPRi parts, selectable markers...

...tag neurons activated by opioid withdrawal with tdTomato and assessed cFos expression. She will explore...periaqueductal gray (PAG), with red neurons expressing tdTomato and green neurons expressing cFos. Image from ...painful stimuli are processed by a network of excitatory and inhibitory neurons whose function and connectivity...where she engineers genetically encoded voltage indicators. Tian developed a video-based high-throughput...archaerhodopsin‑derived genetically encoded voltage indicator which had improved performance in live mouse brains... Currently, she is using the improved voltage indicator coming from the screen to understand mouse cortical...Tian. Figure 5: Citrine-tagged voltage indicator expressed in mouse cortex. Image from He Tian....

...crosslinking agent, a signaling ion, and an allosteric modulator. Because of its importance, scientists have developed... a calcium sensor Genetically encoded calcium indicators (GECIs) are fluorescent proteins linked to calcium...in living cells and organisms. Unlike chemical indicators, these DNA constructs can be delivered to intact...:509-18. Inoue M. Genetically encoded calcium indicators to probe complex brain circuit dynamics in vivo...Kanemaru K, Iino M. Genetically encoded fluorescent indicators for organellar calcium imaging. Biophysical journal...neuronal activity with genetically encoded calcium indicators. Cold Spring Harbor Protocols. 2012 Jun 1;2012...signaling using genetically targetable fluorescent indicators. Nature protocols. 2006 Aug;1(3):1057-65. Resources...

...and helper T (Th) cells. These cells produce inflammatory cytokines - interleukin (IL)-12 and interferon... eliminate early stage cancer cells, chronic inflammatory responses can augment tumor growth. Indeed, ... to precede cancer development, for example, inflammatory bowel disease is associated with the onset of... 1 (CSF-1). Additionally, tumor cells recruit regulatory T cells (T-regs) into their microenvironment ...TGFβ is also known to change CD4+ Th cells into regulatory cells to suppress function. Creating Tolerance...cell-surface proteome to inhibit expression of co-stimulatory molecules needed for T cell effector function...identities based on their level of progression, anatomical location, characteristic (solid localized tumors...

...following: the elevator pitch, talks, the poster session, networking, and social media. The elevator pitch Before... Before the conference Practice your “elevator pitch,” the 30 - 60 second blurb you will use to introduce...work you are familiar with. You can tweak your elevator pitch slightly if you know a bit more about the...or talk. After the conference After using your elevator pitch several times at the conference, you might...might notice some trends. Were there parts of your elevator pitch where the other person seemed confused, ...questions. If you or your lab identifies a potential collaborator from the meeting, reach out to them, pointing...

...development programs for bioscience educators. Using these hands-on tools, educators introduce their students to...a backbone structure of a protein, based on its atomic coordinates. These wire models were useful in that...necessary to download a pdb file of the structure’s atomic coordinates, and “open” these structures with molecular...dangerous. Subscribe to CRISPR Blog Posts How do educators and students use molecular models?   We created...something tangible. Under the guidance of a trained educator, these instances of focused attention become magical... their labs. Soon after we began working with educators in our Center for BioMolecular Modeling, we spun...

...The first is the study of non-coding regulatory elements. Regulatory DNA function is typically inferred ...bloggers! Daniel Bauer is a pediatric hematologist and principal investigator at Boston Children’s Hospital and...interested in exploring the function of non-coding regulatory elements in their native chromatin context. An...PhD student at Harvard Medical School in the laboratories of Stuart Orkin and Daniel Bauer. He received...

...employing optogenetic regulators to initiate the event being monitored. Optogenetic regulators respond to specific...initiation wavelengths, excitation of the optogenetic regulator leads to spurious activation of the FRET sensor...interested in using photoactivatable, optogenetic accuators to depolarize or hyperpolarize membranes. Traditional...wavelengths needed would also activate the optogenetic accuators. The researchers took a FRET reporter and adapted...biosensors that can even be paired with optogenetic regulators. Many of these tools are available from Addgene...2017) Genetically encoded bioluminescent voltage indicator for multi-purpose use in wide range of bioimaging...

...upon a moratorium until another meeting, three branches – a private funder, government regulators, and ...there was a generally agreed-upon international moratorium on utilizing CRISPR/Cas9 in humans. This topic...mtDNA transfer options being investigated; A UK regulatory body tasked two separate non-governmental groups...technology to research centers engaged in long-term, mandatory follow-ups and observational studies of the technique...stop punting decisions on scientific, ethical, regulatory, and general public consensus to the next meeting...

...lipoproteins, shuttles lipoproteins through the circulatory systems, but can also lead to the formation of...as both a powerful tool for generating complex regulatory systems and further the study of natural σ54-... PMID: 31451697. Genetically encoded voltage indicators for use in worms Article contributed by Brook...Brook Pyhtila Genetically encoded voltage indicators (GEVIs) change fluorescence in real time when neurons... excitation light intensities have made these indicators difficult to work with.  The Gottschalk lab has...microbial rhodopsin GEVIs in excitable cells of the nematode Caenorhabditis elegans. They have found several...

...site-specific recombinases, regulatory elements, developmental regulators, and lots of viral components...test many different combinations of promoters, terminators, solubility and purification tags, connectors... examine the effects of different promoters, terminators, enhancers, and cross-cassette interactions in...Standard and New E. Coli Part Library Enable Rapid Combinatorial Design for Synthetic and Traditional Biology... Signaling in CAR T Cells. Cold Spring Harbor Laboratory, 17 Feb. 2024. Crossref, https://doi.org/10.1101...Emerging CRISPR Technologies. Cold Spring Harbor Laboratory, 27 Oct. 2023. Crossref, https://doi.org/10.1016...

...environmental modulation of hepatocellular cancer cell growth." Hepatology 54.4 (2011): 1237-1248. PubMed... was contributed by Bárbara Pinho, a science communicator at the Portuguese science museum "Fábrica Centro...myofibroblasts, neuroendocrine, adipose, immune, inflammatory, angiogenic vascular, endothelial, and stromal...seems to attack us by releasing mitogenic growth mediators and improving cancer cells proliferation.  How... Pinho! Bárbara Pinho is currently a science communicator at the Portuguese science museum "Fábrica Centro...

...you'll find in this post: Tools for labeling excitatory inhibitory synaptic proteins Fluorescent protein...corner New from the viral service Labeling excitatory and inhibitory synaptic proteins with FingRs ...developed AAV vectors to selectively visualize excitatory and inhibitory synapses in the brain.  ...scaffolding proteins PSD95 and gephyrin to label excitatory and inhibitory synapses, respectively. Once expressed... fluorescent proteins targeting inhibitory or excitatory synapses or that are Cre-inducible, researchers... cycle in vivo, particularly if the cells are migratory or in deep tissues. To resolve this issue, Shangqin...

... the notation works? The + operator denotes intersection and the – operator denotes set difference. That...inclusive markers of regulatory regions in the genome, including enhancers, promoters, insulators, and more. I...enhancers, lncRNAs, repetitive elements, repressors, insulators, and more. We know that this matter exists, but...criteria: no TTTTT allowed (this is a polymerase terminator), and no off-target effects for the identified...

...discovered the modular format of some transcriptional activators: that the DNA binding domain (DBD) and transcriptional...of whether it interacts with a transcriptional activator (1a) or a repressor (1b). Scientists took advantage... original Y2H systems utilized the yeast Gal4 activator, bacterial LexA DBD and the lacZ reporter gene...transcription-activating function of a eukaryotic regulatory protein. Science. 1986; 231:699-704. PubMed PMID... and Ptashne, M. A eukaryotic transcriptional activator bearing the DNA specificity of a prokaryotic repressor...PubMed PMID: 2547163. 5. Ozawa, T., Kaihara, A., Sato, M., Tachihara, K., Umezawa, Y.l. Split luciferase...

... concrete happen. You may have more willing collaborators than you realize. Being at a university provides...meetings and take minutes. Get to know your collaborators from other institutions personally on conference...relationship with the perspective of my potential collaborators in mind. Being at a university allows you to...with people at your organization. You can meet collaborators  for lunch, make a point of talking to them ...as messages and keep you up-to-date on your collaborators’ professional developments without directly ...to remind myself to check in regularly with collaborators for example on a holiday or for professional...

...has a great team of moderators who facilitated scheduling, filtered out inflammatory questions, and even...and the moderators were on top of any comments that were at all trollish. Kudos to the moderators for keeping...lists of forums on particular topics. Volunteer moderators set the rules for these forums and try to keep...days). Redditors ask serious questions What the moderators did not delete or suppress were the large number...

...crop species including potato, corn, tomato, wheat, mushroom, and rice (Khatodia et al., 2016). Despite...https://doi.org/10.1128/mmbr.67.1.16-37.2003 Khatodia S, Bhatotia K, Passricha N, Khurana SMP, Tuteja N (2016... al., 2013). Nuclease dead Cas9 (dCas9) based activators (such as dCas9-VP64) or repressors (dCas9-KRAB...

...calcium indicator (Shemetov et al., 2021). In addition to FRET, intensiometric calcium indicators NIR-GECOs...performance, and applications of near-infrared calcium indicators in this recent review (Shcherbakova et al., 2021...encoded near-infrared fluorescent calcium ion indicator. Nat Methods 16, 171-174 (2019). Matlashov, M..... A near-infrared genetically encoded calcium indicator for in vivo imaging. Nat Biotechnol 39, 368-377...Near-infrared and far-red genetically encoded indicators of neuronal activity. J Neurosci Methods 362,...

...expression. Fusing dCas9 to a transcriptional activator like VP64 or VPR activates transcription, whereas... is their persistence and inheritance. CRISPR activators and repressors are thought to be reversible once...cases, epigenetic modifiers may work better than activators/repressors - dCas9-p300 increased transcriptional...enables high-throughput screening for functional regulatory elements in the human genome. Nature Biotechnology...engineering CRISPR toolkit for probing in vivo cis-regulatory interactions in the chicken embryo. BioRxiv: ...Additional Resources on the Addgene Blog Cas9 Activators: A Practical Guide CRISPR Between the Genes: ...

...have begun to use more precise genetic tools: activators, inhibitors, sensors, recombinases, and more....which was recently published in Frontiers in Neuroanatomy.  Here are the three main takeaways from the...achieved in nontransgenic animals by using cis regulatory elements such as promoters and enhancers, multicistronic...multicistronic vectors, and post-translational regulatory elements. Examples of these approaches are given...specificity of the targeted cell population.  Combinatorial strategies can also be used for retrograde tracing...

...cause the least amount of immune response. Any investigator who is using an in vivo animal model should ...identified with AAV2 (5), AAV6 (6) and AAV5 at excitatory connections (3). These facts beg the question...by the use of AAVs? 1) Combined functional and anatomical mapping of circuits In our lab, we use AAV-DREADD...these complexes are so robustly expressed that anatomical assessments can be easily performed too. While...identified this feature of AAVs as a new tool for neuroanatomical tracing (11). With the ability to produce neuron... Floris G., et al. "A fourth generation of neuroanatomical tracing techniques: exploiting the offspring...

...Hendel et al. 2015). Low detection rates mean the investigator can be relatively certain that their samples...off-target mutations are essential data points for investigators looking to definitively link genotype and phenotype...viable options for multiplex sequencing in many laboratories. Nevertheless, unbiased detection isn’t as sensitive...genome-wide unbiased methods. However, most investigators use single cell clones for in vitro CRISPR experiments... Hough is a Biochemistry PhD candidate in the laboratory of Prof Steve Jackson at the Gurdon Institute...Resources on the Addgene Blog Learn to Use Cas9 Activators Perform Genome-Wide Screens with CRISPR Generate...

...professional-grade design software, like Adobe Illustrator, and I didn’t want to pay for these programs ... Vectr is like a pared down version of Adobe Illustrator. There’s both a web and a desktop version of ...Inkscape Inkscape is the most similar to Adobe Illustrator out of the three options for drawing your own...20,000 icons is designed by professional medical illustrators with input from life scientists. It’s easy to...medical images organized into four main categories: anatomy and the human body, cellular biology, medical specialities...

...CsChrimson::tdTomato, an optogenetic tool (CsChrimson) attached to a fluorescent indicator (tdTomato), as the...intermediate (d), and sparse (e) labeling of the calcium indicator, GCaMP6f, in the Drosophila optic lobe. Image ...dendrites of T5 neurons by placing the calcium indicator GCaMP6f in the effector position, as well as to...

...same page. Ideally, these meetings include all collaborators, from the lab members who are conducting the...interesting results or ask questions to your collaborators. This helps everyone feel included in the project...frequently the entire group will meet. You and your collaborators should then hold each other accountable to these...and circumstances permit, think about having collaborators spend time with each other in person. One possibility...workshops are another great place to meet up with collaborators. Being able to present data from multiple aspects...

...pAAV-Syn-FLEX-rc [Archon1-KGC-GFP-ER2] pAAV-FLEX-tdTomato pAAV-GFAP104-mCherry pAAV-mDlx-NLS-mRuby2 CAG-... also be used in conjunction with optogenetic activators and inhibitors that respond to blue light. We...fluorescent-tagged molecular tool.   Find pAAV-FLEX-tdTomato, pAAV-GFAP104-mCherry, pAAV-mDlx-NLS-mRuby2, CAG-NLS-GFP... pAAV-hSyn-DIO-mCherry 59462  AAV2  pAAV-CAG-tdTomato (codon diversified) 37825  AAV8*, AAV9*  pAAV-CAG-GFP...Also coming in our new 20 ul trial size! Voltage indicators Plasmid Serotype Name 119036  AAV1  pAAV-hsyn-flex-Voltron-ST...

..., the researchers did not have access to a P3 laboratory (BSL-3), a high-security facility needed to perform...  Figure 2: When a researcher needs a collaborator for a specific task, Crowdfight will: 1) Find...researchers, such as postdocs and principal investigators. They also come from many different scientific...website. Once curated by our in-house team of collaborators, the requests were forwarded to our volunteer...Biomédica de Salamanca (IBSAL) and one of the coordinators at Crowdfight. She is currently based in Spain...

...effect of mutations of these regulatory elements on gene expression. The regulatory element (wild-type or mutant...gene, such as luciferase, usually serves as an indicator of transcription within cells, where detection... most often used to investigate the effect of regulatory elements, such as promoters, enhancers and untranslated...can then be used to quantify the effect of the regulatory element. Depending on the specific luciferase...

...her then was this: Can you engineer CRISPR-Cas9 activators that work well enough on any gene that they could...which they call CRISPR/Cas9 Synergistic Activation Mediator or SAM. SAM is an engineered protein complex for...information from the structure to guide and improve activator design,” she said. “Before it was a little like...Konermann recognized as an ideal spot to fuse the activators instead of at the protein’s C terminus. With ...repressing or inducing individual transcripts. The activator (CRISPRa) sgRNA library uses the sunCas9 system...

...human pluripotent stem cells: a transcription activator-like effector nuclease (TALEN) kit consisting ...Cell Stem Cell paper, for example, we generated hepatocytes to examine the effect of a gene on cholesterol...Addgene: Tell me about the history. How had investigators done this kind of work before? Musunuru: In ...Unfortunately, it is time consuming and difficult for investigators without a lot of expertise to create zinc finger...and puts it in the hands of pretty much all investigators. Our kit makes it extremely simple to design...

...generation of human hepatocyte-like cells from induced pluripotent stem cells. Hepatology, 2010. 51(1): p....of these cell types [13]:  Germ cells [14] Hepatocytes [15] Pancreatic β-cells [16] Intestinal tissue... intermediate pluripotent state is not always obligatory in cell conversion processes [35]. Rather than...transdifferentiation. Multiple cell types such as fibroblasts, hepatocytes, and pancreatic exocrine cells have been successfully..., A.E., et al., Directed differentiation of hematopoietic precursors and functional osteoclasts from human...

...Schmidt, a Marie Curie Fellow at The Sainsbury Laboratory. Are you a science student or early career researcher...in education advocacy non-profits like Phipps Conservatory or the Brilliant Club and share your knowledge... photo competition. Or nominate yourself as an educator for a short, animated TED-Ed lesson. 6) Network... pros Where do you meet like-minded science communicators? Attend the annual flagship meetings of AAAS...bananas as a Marie Curie Fellow at The Sainsbury Laboratory. She is passionate about science communications...

.... The first was a new class of programmable riboregulators, called toehold switches, which allow for the...allowed gene circuits to be deployed out of the laboratory in a cell-free and biosafe format that was stable...circuit-based sensors to be used outside of the laboratory. The detection threshold for these RNA sequences...the technology through product development, the regulatory process, and manufacturing so that we can get...., et al. "Toehold switches: de-novo-designed regulators of gene expression." Cell 159.4 (2014): 925-939...