... Career Planning Resources. The key to ensuring success during graduate school and beyond is to live in...transferable skills while working in the lab and by offering advice on areas where you might need some help...on what I have found to be especially beneficial during my own graduate career – and in fact, some of the... a job fair! Similar to a conference, job fairs bring you face-to-face with working professionals, with...the ACS career fair, which are larger meetings offering resources for students seeking employment within...graduate schools will even pay for you to attend!), exploring a job fair, and utilizing your local career development...

...well. Making the transition from the bench can be jarring for the unprepared, and unfortunately, there aren...who referred to her lab as her family three times during a 10 minute conversation. Leaving a tight-knit ... “I’m a scientist.”    What should you expect during the lab → office transition? I’ve already provided...sure, but it also eliminates that feeling of floundering one can have when starting in an academic lab...science,” and virtually every person you talked to during the day was also a scientist. But now you also ...upsetting, there are ways to get back to the bench during your off hours. DIY bio labs are cropping up in...organizations like Science from Scientists, which try to bring real scientists into schools to provide students...

...editing in rice. In Genome Engineering: The CRISPR-Cas Revolution, Cold Spring Harbor Laboratory (2018)....et al., 2016) invented CRISPR-base editors by tethering cytosine deaminase with Cas9 nickase (nCas9). ...to an adenine base editor. A massive protein engineering effort by Gaudelli et al. resulted in the successful...it with dCas9, and transformed it into E. coli harboring the defective CamR gene. Screening for surviving...colonies and subsequent rounds of evolution and engineering gave rise to a mutant TadA (TadA*) that satisfactorily.... Kang, Beum-Chang, et al. "Precision genome engineering through adenine base editing in plants." Nature...

...Ellis does research in synthetic biology and bioengineering including a recent effort to construct a synthetic...Synthetic Biology and Innovation - A shared space for bringing synthetic biology to the UK with foundational ... SynBio. Chapter 3: Synthetic biology as the engineering of biology at the level of DNA using modular,...in the Ellis lab including yeast and E. coli engineering, microbial cooperation, and antibiotic production...lectures synthetic biology for the Department of Bioengineering. He graduated from Oxford University and then...supervision of Prof Jim Collins and specialising in engineering gene regulation in yeast. Dr. Ellis is now one...

...Addgene has done a really good job at figuring out how to operate during the pandemic, but it’s important ... addition of Cicalese to the Board of Directors brings human resources experience and insight to the now... the near future? Right now it’s a matter of maneuvering through the impact of COVID-19. I think Addgene...which raises questions like “How do you start to bring people back to on-site work?” and “How do you start...not working so that I’m available to spend time caring for my mother. For fun, I enjoy pilates, jewelry...

...other blog posts on sharing science: 10 Ways to Share Your Science More Scientific sharing posts! ...Here at Addgene, we’re dedicated to advancing and sharing science! In association with the Harvard graduate... a wide variety of programs, including fall and spring lecture series, science cafes, and Reddit AMAs.... sharing... Mary Gearing...

...you for inspiring us with your breakthrough research and your commitment to scientific sharing. In the... of plasmid sharing  Interested in more facts about Addgene's 10 years of plasmid sharing? Check out this...for mammalian gene silencing. TALENs - Genome engineering methods improved rapidly in 2011, from zinc finger...continue to improve our services and work to make sharing even easier. We can't wait to see what you put ...

...mapping to investigate DNA sequence content by comparing the fragments created by different restriction...breakthrough for determining the order of nucleotides in a string of DNA (7). Sanger sequencing uses modified nucleotides...become obsolete. For instance, the popular genome-engineering tool, CRISPR, may one day make techniques used...DNA strand as it slips through a tiny pore by measuring differences in ion flow caused by the different...under $1,000 (10). The future of sequencing should bring efficient, low cost methods for sequencing large... as both time and research progress. These will bring faster, higher throughput, and potentially more ...Andrew Cho, and Ashok B. Kulkarni. "Overview: engineering transgenic constructs and mice." Current Protocols...

...Methods for measuring Dopamine In vivo microdialysis The gold standard method of measuring DA and other...someone is awake, agitated, or asleep. Though DA firing patterns (or the frequency of its release) are ...brain slices Visualization of DA release dynamics during locomotion in freely moving mice Record DA dynamics... in live mice Report modulation of DA signaling during learning and reward consumption in live mice Find...of our viral vectors blog posts Find tips for preparing AAVs Download our viral vectors 101 eBook Resources...

... Kyle Hooper at Promega. Researchers have been sharing plasmids ever since there were plasmids to share...applications of the Enhanced Nano-Lanterns including monitoring multiple cellular events such as the dynamics...protein binds to calcium it changes conformation and brings the two fluorescent proteins together thereby increasing... blue-shifted fluorescent protein (donor) and measuring the resultant emission from the red-shifted fluorescent...release with an optogenetic receptor, melanopsin, requiring brief exposure to 470nm blue light. To avoid overlap...is available through Addgene (Plasmid # 87127). Pairing NanoLuc® luciferase with fluorescent proteins has...in vivo optical imaging and flow cytometry for monitoring tumorigenesis. Cancer Res. 75, 5023-33. PubMed...

...attB-attP pairs on a register to record all permuted substrings of a set of chemicals. Specific examples of arrangements...arrangements that should be able to record all permuted substrings of up to seven chemical inputs are given in Table...50% GC content) so that individual parts and/or strings of parts may be PCR-ed out of their current registers...population to correctly record each possible permuted substring of the inputs, and that five hour inductions were...cells to correctly record each possible permuted substring of inputs (see Figure S9 of our paper (1)). We...al. "Serine recombinases as tools for genome engineering." Methods 53.4 (2011): 372-379. PubMed PMID: ...Check Out More Lu Lab Plasmids Find More Genome Engineering Tools  ...

...biology and brings 10 years of research experience with stem cells to Transposagen's cell engineering team. ... sequence, making it very useful for genetic engineering approaches. Most importantly for this technology...selection cassette can be incorporated, and used during the differentiation process. Moreover, a splinkerette-PCR...Craig, “piggyBac transposase tools for genome engineering.,” Proc. Natl. Acad. Sci. U. S. A., vol. 110,...Sleeping Beauty Transposase Read up on Genome Engineering with FLEx Vectors  Learn how Homology Directed... genome engineering...

... shift toward hiring and promotion practices that focus on reproducibility and ensuring the health of ... at Harvard University 29:31 - 49:45 - Reagent Sharing - Susanna Bachle, Addgene the nonprofit plasmid...of different types of replication in Nosek and Errington 2017 and Schmmidt 2009, but for our purposes we...experiment wrong. However, even simple procedures like pouring a plate or running a gel have intricacies that ...science Twitter is very active with many scientists sharing their unpublished data. There are blogs - both ...novelty and impact. Journals could also begin requiring reproducibility checklists that at least make ...

...from Addgene. How Addgene is Reimagining Antibody Sharing Addgene is building our antibody resource, which...community-building. The transparency begins with sharing sequence information for the antibodies in our ...accompany our antibody collection.  Addgene is sharing our protocols for recombinant antibody production... antibodies for your research. Please consider sharing your experiences with antibodies from Addgene via...other. Together, we can transform the antibody sharing landscape as well.     References Andrews NP, ...a nonprofit that has been supporting hybridoma sharing within the community for decades. Educational Resources...

...We’re big fans of all forms of research sharing and with Open Data Day 2020 coming up on March 7th, what... what better way to celebrate open data sharing than with Addgene’s AAV Data Hub! We launched this pilot...ranks of our growing group of Addgene AAV data sharing champions? If you’ve recently used Addgene AAV ...simple steps you can take to input your data for sharing on the site. The entry form has 5 general question...questions and so that we can give you recognition for sharing your data. Materials: What virus and serotype did... have any feedback at all, we’d appreciate you sharing it through this form. In honor of Open Data Day...

...fluorescent proteins were best imaged after tissue clearing with SeeDB2 (Ke et al., 2016, 2018).   How to...amount of tTA to achieve highest expression levels. Clearing large tissues - Fluorescent proteins are very ...with ScaleCUBIC1 (Susaki et al., 2014), before clearing with SeeDB2. For more information, visit our ...action, watch the video below depicting dendrite wiring of mitral cells.   Many thanks to our guest... of neuronal circuitry with an index-optimized clearing agent." Cell reports14.11 (2016): 2718-2732. PubMed...26972009. Ke, Meng-Tsen, and Takeshi Imai. "Optical clearing and index matching of tissue samples for High-...

...vector into the culture medium during adeno-associated virus manufacturing." Human gene therapy21.10 (2010...Enhancement in vector stability." Biotechnology and bioengineering 94.2 (2006): 322-329. PubMed PMID: 16528756...pressure and sodium butyrate." Biotechnology and bioengineering 42.5 (1993): 601-610. PubMed PMID: 18613082...and antibody production." Biotechnology and bioengineering 37.10 (1991): 989-993. PubMed PMID: 18597326... Kamen. "Hyperosmotic pressure on HEK 293 cells during the growth phase, but not the production phase,...

...reagent-grade endangered Martian chicory root, but considering this is a needle in a haystack type of objective...In retrospect I used a way too high Cas9 MOI considering that A549 cells are relatively easy to transduce...could avoid potential off target artifacts by comparing the phenotypes of multiple gRNAs. Targeting different...lanes 0.3 % 3.0, Figure 5, lane 2) is faint, so comparing it to the undigested control (Figure 4, lane 0...straightforward, and is a testament to how far genome engineering has come. For comparison, in 2007 it took about...for Designing Primers UCSC Genome Browser: For eploring genomic DNA ImageJ: Free Software for Analyzing... genome engineering...

...ranges from astronomy to bioethics to chemical engineering and everything in between. Plus, there are many...pursue it. It can manifest via research and data gathering, regulatory efforts, policy implementation, program...societies host “Hill Days” where they train and bring researchers to Congress to advocate for increased...normal!  Action Items:  Start a career journal during your current job and note what makes your heart...writing and presenting for non-scientist audiences during your time in graduate school through outreach organizations...working in student government, and teaching and mentoring others (see more in Figure 2). Starting early ...programs can connect you with alumni before and during your application process so you can learn more!...

...translation is key to cellular function, especially during development or stress. With ribosome profiling,...to track translation in two different ways: by monitoring the first round of translation or by tracking...NLS-MS2-RFP) the reporter mRNA, which contains the tethering sites for the fluorescent proteins and is driven...double labeled, indicating translational repression. During later stages, single RFP-labeled osk and Oskar ...with the red reporter mRNA. Yan et al. used harringtonine, a translation inhibitor that stalls ribosomes... Mary Gearing...

...how it works. I used to stay up past my bedtime, poring over my grandparents’ medical textbooks by torchlight... I asked participants to read short abstracts comparing the mortality rates between two controversial ...and biased. As a result, the control abstract comparing two fictional medical issues – chronic and acute... Kahan et al., 2012). Scicomm in the community During my PhD, I’ve also found opportunities for more ...community. I brought along printed copies of studies comparing the filtration efficacy of different materials... in technical jargon that even scientists in neighbouring disciplines might struggle to understand. Why...

...Varmus H. Retroviruses. Cold Spring Harbor Laboratory Press; Cold Spring Harbor, NY, USA: 1997. PubMed...them attractive and tractable tools for genome engineering. Scientists have engineered viruses to target...incompetent viral particles, are required. We will be covering these safety measures in a separate, upcoming ...breakdown of the nuclear envelope, which occurs during mitosis. The most well-known retrovirus is HIV....PubMed PMID: 21433340. 4. Cronin J, et al. “Altering the tropism of lentiviral vectors through pseudotyping...

...educational content Addgene truly believes that sharing speeds science, and our entire mission is to enable...informative content to help researchers in creating and sharing knowledge, related to the types of materials we...help.addgene.org, where you can easily search articles answering all of our frequently asked questions!    ...published alongside our new antibody services in the spring of 2022. The first step was to understand information...licensed and cited figures from papers or image sharing repositories. Then we format, do a final review... and knowledge changes and require updating or retiring. CRISPR/Cas9, for example, has gone through rapid...

...to keep their cargo below 4.4kB (with the ITRs bringing total genome length to ~4.7kB.  If your insert... kB, then you would be oversized even before considering the need for some manner of polyA. In this scenario...intein approach.   Q3: What’s the best method for titering AAV? Is there a difference in titer results between...equal animal dosing, we advise that users keep titering method consistent across samples within a project...microglia in vivo.   Q5: What is the efficiency of co-delivering different AAV particles into the same cells in...any difference in transfection efficiency when delivering these viruses via retro-orbital vs. tail vein...

...introduced by lambda red recombineering and Multiplex Automated Genome Engineering (MAGE) will always be ...methods for bacterial genome engineering   Learn more about lambda red recombineering of bacteria Use site directed...within a defined region of a genome. Require stringent bacterial growth conditions. Expression of an ...only increasing the standard mutation rate seen during DNA replication by 120-fold over background. Due...

...and Keira Havens, co-founders of Revolution Bioengineering. Read their previous blog post about how they...directed at GM crops has burdened this emerging engineering technology. Monoculture and chemical spraying... you have developed a genetic tool for plant engineering, please share it with Addgene to both build the...biology community and to support more efficient engineering of a better tomorrow. Thank you to our guest...outside of Washington D.C. and has always enjoyed exploring the outdoors and experiencing nature first-hand...SynBio Reading: Learn more about Revolution Bioengineering Follow RevBio on Twitter Like RevBio on Facebook...

..., too. And remember, if you discover during grad school or during your postdoc that you enjoy and have...engineers were attending the same meetings and hearing the same words, they were leaving these meetings...know what kind of plate they were talking about during the meetings. And “plate” certainly wasn’t the ...experimental protocols. Clarify: Asking for clarification during a meeting can help get everyone on the same page...have come upon any other effective approaches for clearing up communications lines in your professional life...

...or little deterioration or change of the signal during the course of the experiment. The best fluorescent...photobleaching rates (Shaner et al, 2008). This brings us to the key question: what is the best way to...photostability of different fluorescent proteins? Measuring photostability Since it is clear that a photostable...measurements for different fluorescent proteins and by comparing how the fluorescence intensity changes over time...than predicted by these high power experiments. Measuring photostability at realistic powers will provide... but they do not mimic the natural situation. Measuring photostability in living cells provides a much...

...Challenges of engineering channelrhodopsins There are three main challenges to engineering channelrhodopsins...channelrhodopsins with high-throughput proteins engineering methods, like directed evolution, because channelrhodopsins...-stimulate” their VTA dopaminergic neurons by triggering a burst of light that could activate ChRger2 ... require fiber-optic cannula implantation, but altering ChRgers so they can be activated with tissue-penetrating...2019) Machine learning-guided channelrhodopsin engineering enables minimally invasive optogenetics. Nat ...

...replication of virus and its infectivity in check. Engineering controls, safety systems that are built into ... you can receive appropriate treatment and/or monitoring. Virus-specific concerns   In nature, some viruses...protection! When centrifuging viruses (a necessary step during production), there is a significant risk for aerosol...of following your lab’s biohazard policies. Administering Virus Many virus experiments involve needles...accidents is not following safety protocols.) If administering virus to mice, part of your preparation should...let you work with these valuable tools without endangering yourself or others. Happy transducing! Resources...

...Here's what you'll find in this post: Epigenetic engineering with single-pot assembly Tracking cell-cell interactions...New items from the viral service Epigenetic engineering with single-pot assembly By Eric Perkins Though...epigenetic researchers custom design their own engineering plasmid tools in about a week. Taking advantage...receiver cells. What’s more, G-baToN can be used to bring cargo, such as protein or DNA, to manipulate function...CRISPR Plasmids and Resources page.  Plasmids for delivering CRISPRi components by a single AAV vector are...Choudhary's lab developed a chemogenetic system that brings Cas9 to ubiquitin ligase for degradation.    ...

... the abdomen. They are a popular choice for administering different drugs and viral vectors. Like IV injections...suggests, in the muscle. Common uses include administering vaccines, pain meds, or other drugs. Identifying...don’t always require needles. If needles make you cringe or your experiment will allow you to avoid poking...straight to the stomach. This technique involves administering medication through a feeding tube with a bulb...introduction to common injection routes in mice! When considering what method you’d like to use, remember that ...Organisms Additional resources on Addgene.org Genome Engineering Guide CRISPR Mammalian Expression plasmids Cre-Lox...

...tissue clearing Similar to light microscopy, there have been significant advances in tissue clearing techniques...brightness with increasing depth caused by light scattering and absorption.  Finally, the photomultipliers...techniques during this century.  The first successful attempt to render fixed anatomical preparations transparent...Ultramicroscopy with tetrahydrofuran-based tissue clearing (13,14). A few years later, Karl Deisseroth’s ...  The combination of LSFM with multiple tissue clearing strategies and neuronal labeling methods will ...

...by loss of protein during digestion, or masking of epitopes during sample anchoring and gel polymerization... Tillberg, et al. 2016). That being said, the anchoring, digestion, and expansion steps can impact the...occurs, due to a reduction in target concentration during expansion. One group has found that using Fab fragment...et al (2023) Magnify is a universal molecular anchoring strategy for expansion microscopy. Nat Biotechnol...

...where droplet digital PCR (ddPCR) comes in. AAV titering using quantitative PCR Before we dive into the... PCR is complete, the results are analyzed by comparing the threshold fluorescence of the viral DNA to...reasons, we are in the process of transitioning our titering method from qPCR to ddPCR. Droplet digital PCR...reference, meaning saving on reagents (and time)! AAV titering using droplet digital PCR Droplet digital PCR ...generator, you will have to be careful about transferring your droplets from the droplet generator to ...detection of microbes. Some applications include measuring viable probiotics and detecting circulating pathogens...

...held. Imagine this situation: You’ve spent hours scouring a company’s LinkedIn pages, given yourself the...guaranteed to get the job, however, many times the hiring team will also want to examine not just technical...technical skills, but also transferable skills, before offering someone their dream job; They want to know if ... and to demonstrate them to the people who make hiring decisions. Though having all the technical skills...get a great snapshot of the skills that you can bring into pretty much any other workplace. To get your... for non-academic science careers Learn about mentoring Resources on Addgene.org Check out our careers...

...Knight TF. Engineering BioBrick vectors from BioBrick parts. Journal of biological engineering. 2008;2:5... fluorescent protein were fused in-frame to a tethering partner, which directed localization of the fluorophore...Construct Fluorophore Excitation/emission maxima Tethering partner Subcellular localization 1 TagBFP 399nm...Constraints on the order of the modules, because neighboring fragments require compatible cohesive ends Engler...biological part assembly. Journal of biological engineering. 2010;4(1):1. Pubmed PMID: 20205762. PubMed Central...

...the non-radiative transfer for energy between neighboring fluorophores, FRET can be used to detect the ...ratios) to generate useable data. What’s more, measuring and quantifying FRET is often easier said than...complete FP because the PIPs they’re tethered to bring them into close proximity (Figure 1). Formation ...independent of PPIs. Overexpression can be assessed by comparing the expression levels between BiFC constructs ...Analysis of Protein Interactions in Live Cells. Cold Spring Harb Protoc 2013:pdb.prot076497. https://doi.org...Fluorescent Protein 101 blog posts Read our blog post covering an introduction to FRET Resources on Addgene....

... parents in STEM careers, chances are they’re wondering how they could contribute to the school, too. ...Middle school: supplementing the science curriculum Sharing my expertise with our district middle school was... an authority in this area. What is genome engineering? How does CRISPR work? This section was essentially... of the talk by running it by the teachers and sharing some slides on Twitter, where I was able to get...computers and the internet before it, genome engineering technology is moving along much faster than it...understanding genetics is so timely.” Genome engineering, and many other technologies and fields in biology...

...optimization? Codon optimization is an approach in gene engineering to improve gene expression by changing synonymous...efficiency and thus protein expression without altering the sequence of the protein. As DNA synthesis ...tRNAs, including those that exhibit wobble base-pairing (a tRNA that can recognize multiple synonymous ...of translation. The ribosome may slow and pause during elongation which may actually be necessary for ... SA, Zhou W, Mauro VP (2005) An mRNA-rRNA base-pairing mechanism for translation initiation in eukaryotes...02)00362-x Stadler M, Fire A (2011) Wobble base-pairing slows in vivo translation elongation in metazoans...

...But CRBN has been difficult to study in vitro, requiring either expressing it with additional cofactors...faster, with expression levels comparable to GFP during early embryogenesis (Figure 3). mScarlet-I3 also... through the middle of each embryo are shown, comparing GFP, wrmScarlet (mScarlet), and the new mScarlet3...proteins of interest) and mScarlet-I3 for scenarios requiring rapid maturation and prolonged imaging with minimal...high rate of spontaneous antibiotic resistance interfering with selection. Using an inducible Cas9 expression...to cause disruption of the target gene. Lastly, curing the mCherry-sgRNA plasmid leaves an edited strain...

... You need to balance respectfully answering questions while steering the conversation back to your vision...proposal or a 10-year research plan. I suggest preparing for both types. Your research vision is your approach...proposal will distinguish between competing models. Comparing model A vs. B is more constructive than testing...vision. On the day of your chalk talk Bring your own fresh set of erasable markers. You’ll have the colors...and consider eating one before your chalk talk. Bring your own bottle of water. If possible, write down...

...the Wyss Institute for Biologically Inspired Engineering. CRISPR/Cas9 is an enormously plastic tool and...activator by simply providing a new gRNA rather than engineering an entirely new protein. This makes it much cheaper...types of Cas9 activators, check out our blog post covering VP64, Suntag, SAM, and VPR. Worry less about off...the Wyss Institute for Biologically Inspired Engineering.     Alejandro (Alex) Chavez is a member of the...develop several popular CRISPR-based tools for altering DNA sequence or regulating gene expression.   ...Church GM (2015) Unraveling CRISPR-Cas9 genome engineering parameters via a library-on-library approach....

...inteins. Split inteins are a pair of naturally occuring polypeptides that, when at the ends of two proteins...Marraffini LA, Zhang F (2013) Multiplex Genome Engineering Using CRISPR/Cas Systems. Science 339:819–823...Spinner HB, Baney KLM, Chuck J, Tan D, Knott GJ, Harrington LB, Al-Shayeb B, Wagner A, Brötzmann J, Staahl..., Grimm D (2014) CRISPR/Cas9-mediated genome engineering: An adeno-associated viral (AAV) vector toolbox...Gao L, Makarova KS, Koonin EV, Zhang F (2019) Engineering of CRISPR-Cas12b for human genome editing. Nat... Mary Gearing...

...are syringe or vacuum infiltration. Syringe infiltration (D'Aoust et al., 2009): Usually a syringe without...contains a plasmid with a T-DNA (transfer-DNA) region. During infection this T-DNA region is transferred into...transformation and gene expression can be tracked. During a transient leaf transformation, the engineered...Transient expression of antibodies in plants using syringe agroinfiltration." Recombinant proteins from plants...

...about the particular audience and venue before tailoring each presentation appropriately.  Find out who...many non-scientists be present?  Always ask the hiring manager or host for topic advice. An added bonus...figure out how to make it suspenseful. 4. Start preparing your talk early - like today Don’t wait until ... all graduate students and postdocs form peer mentoring groups to practice giving talks and getting feedback...always have a few extra slides in their back pocket. During your practice rounds, encourage your listeners ...down it is a great segue to talk about how the sharing model works. See if you can elicit helpful comments...

...briefing, to quick notes, to finances, to flights & ordering food - everything is covered.   So why are we ...folders in complex ways, there are solutions that bring more to the table in terms of data traceability ...researchers today generate vast amounts of digital data during their work. It is already becoming impossible to...to keep all their data in one place and avoid scattering intellectual property across various software...functionalities include comments, notifications, sharing files, assigning team members to certain tasks,...compliance requirements – e.g. 21 CFR part 11, HIPAA? Answering this question helps you evaluate whether the ELN...

...interested in measuring a population based on a fluorescent stain, you might be wondering which second ...types of scattering that occur when a cell is pulsed by the laser. Within each type of scattering, you also...to keep it short though, promise! FSC and SSC: scattering To digitally extract your cells based on their...cells stain positively for both markers. When comparing two markers simultaneously, we commonly describe...

...important information for anyone considering a postdoc. In “Changing Labor Laws Bring Increased Postdoc Wages,...readership with over 75,000 views. Changing Labor Laws Bring Increased Postdoc Wages Our most popular blog post...Grad School Advice Part 2: Building Community Engineering the Plant Genome Using CRISPR/Cas9 Healthcare...Reddit for Science Communication Changing Labor Laws Bring Increased Postdoc Wages I hope you all have a great...

... what challenges remain in materials sharing, information sharing, and research reproducibility while ...service and support.  Alongside our material offerings, in 2022 we also rolled out an updated AAV Data...around the world can upload AAV validation data, sharing what works and, equally importantly, what doesn...commitment to supporting the research community through sharing science and distributing high-quality materials...