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...chromosomal DNA.  Plasmids are said to be under stringent control when they depend on the host cell’s initiation...consider the replication mechanism of plasmids under stringent control, such as pSC101. DNA replication and regulation...replication for the bacterial chromosome and for stringently controlled plasmids. Binding of IHF (green) redistributes... cell growth or even kill the host cell. For stringently controlled plasmids, replication is tightly coupled...number.  Let’s consider pSC101. This plasmid is stringently controlled and has a low copy number. Like the... necessary. Iterons are not the only tool to stringently regulate plasmid replication. For example, the...Comparison of replication regulation mechanisms of stringently controlled plasmids pSC101 and ColIb-P9. pSC101...

...also a story of community, cooperation, and sharing. Sharing this story is a truly special way to celebrate...As we celebrate twenty years at Addgene, we are sharing stories of our repository’s positive impact on ... that developed CRISPR tools were committed to sharing them with the rest of the community. They did this...the usefulness of popular CRISPR tools is both enduring and rapidly recognized.   Where is the CRISPR ...appropriate MTAs and customs forms. Of course, ordering the plasmids is the easy part; using them can ... resources on the Addgene blog Twenty Years of Sharing Science  ...

... of proteins without the challenges typically occurring with a fluorescence-based approach. These challenges...vesicles (EVs), edited transcripts could endow neighboring cells with pro-oncogenic traits, facilitating...EGFR. This method offered kinetic, non-invasive monitoring of receptor dynamics at the cell surface. Find...HiBiT-EGFR and EGFR-HiBiT Fusion Vectors at Addgene! Measuring messengers: growth factors Growth factors present...) utilized HiBiT to address these challenges, exploring the binding kinetics of the VEGF-A and the VEGFR2...

...preparation steps of production, purification, and titering. Our newest viral vector service, AAV Packaged...

...Addgene blog Twenty Years of Addgene Sharing: CRISPRTwenty Years of Sharing: Addgene's Viral Vector Service...many countries, import and export regulations, differing customs processes, and the distances the packages...specific to that country. ‌For example, when we began offering temperature-sensitive materials, we noticed a ...shipments arrived in India at room temperature. Partnering with our distributor allowed us to ensure that...shipments weren’t able to clear import control. Partnering with a distributor allowed us to share materials...scientists in China. They were also able to help with sharing information through the use of popular Chinese ...solutions aren’t enough to ensure accessibility, partnering with distributors who have expertise in a specific...

...imagination of most of the authors during this period.  Genetic Engineering Era: Pre-CRISPR  Having visualized...associated with genetic engineering in the recent SciFi movies. Rampage (2018), starring Dwayne “The Rock” ...California, San Diego. The revolution in genetic engineering techniques is a speculation of yesteryear which...but the common themes that these authors were exploring were space and robots. Warping to stars in space...devise methods to manipulate it. The term genetic engineering itself is attributed to SciFi writer Jack Williamson...s Island. In 1976, the world’s first genetic engineering company, Genentech, was founded.  This commercialization...these corporations would extrapolate genetic engineering to exploit human beings. Ridley Scott directed...

...2012). This made them an appealing target for bioengineering, but it wasn’t clear whether they could 1) ...bodies We have shown that all three of these engineering objectives are possible in our recent paper (...from This Publication at Addgene 4. Petroni, G., Spring, S., Schleifer, K.-H., Verni, F. & Rosati, G. Defensive...

...cycle for titering as this will better represent future experiments. The aforementioned titering options...in the long run. Read on for an overview of the titering options as well as the benefits and drawbacks ...measurements via ELISA and qPCR for viral RNA. Measuring p24 levels in a viral prep is straightforward ...transfer plasmid was used in the transfection; if preparing several different lentiviruses in parallel, this...should be considered. Similar to the p24 assay, titering via measurement of viral RNA can overestimate ...viral particles. One of the si mplest methods of measuring functional titer uses FACS to count the number...antibody staining. Check Out Our In-Depth Fluoresence Titering Protocol Another popular approach utilizes the...

...of modified Ti plasmids during plant infection, as well as plasmid yield during preparation in E. coli....the plants that we grow and eat, and genetic engineering will continue to improve plant nutrition, yield..., and pest resistance. Much of plant genetic engineering revolves around Agrobacterium tumifaciens. Agrobacterium...Agrobacterium to ensure that they are maintained during co-cultivation with the target plant species. Thus...with biolistic methods, making it useful for engineering plants that are not susceptible to Agrobacterium...customizability, pSiM24 can be used not only for plant engineering and biotechnology applications, but also for ... plant genetic engineering...

...these defenses. For some applications of genome engineering - such as certain forms of gene therapy - avoiding...tool and an alternative to CRISPRs for genome engineering whose use is only likely to grow in the coming...  genome engineering...

...customers. Details about our production protocols, titering methods, and quality control are described below... or through collaboration with viral vector manufacturing facilities, such as the University of Pennsylvania...are then aliquoted and stored at -80 °C. Titer Titering is either performed by Addgene or by the University...University of Pennsylvania Vector Core. In general, titering is performed by the facility that produced the...those achieved using the standard qPCR method. Titering by the University of Pennsylvania Vector Core ...Purity Purity of AAV preparations is assayed by comparing the relative stoichiometric ratios of the viral...aliquoted, frozen, and stored at -80 °C. Titer All titering is performed on lentiviral preparations that have...

...nuclease fusions Related Genome Engineering Technologies TALEN Engineering Reagents CRISPR Plasmids...Find reagents for engineering and expressing zinc finger proteins deposited by Zinc Finger Consortium...have deposited at Addgene various reagents for engineering and expressing zinc finger proteins for distribution...reagents are provided below: Zinc Finger Genome Engineering Reagents Zinc Finger Arrays for Zebrafish Genes...fingers targeting specific genes Oligomerized Pool ENgineering (OPEN) Reagents - Engineer zinc finger arrays...arrays via OPEN method Modular Assembly Engineering Reagents - Engineer zinc finger arrays via Modular Assembly...

...included continuous outflow of phage, increasing stringency and promoting survival of only the broadest-targeting... Mary Gearing...

...stably integrates into the genome and is propagated during normal DNA replication and cell division. Mobile-CRISPRi... these plasmids for transfer to recipient cells during a tri-parental mating scheme. Mobile-CRISPRi integrates...was variable. They measured transfer rates by comparing the number of colonies that grew on a selective...about other CRISPR methods for bacterial genome engineering   Learn about human CRISPRko, CRISPRi, and CRISPRa...

...of our viral vectors blog posts Find tips for preparing AAVs Download our viral vectors 101 eBook Resources...

...-2, but with many academic research labs closed during the pandemic, it’s hard to get these reagents. ...available through a repository, they remain accessible during these times. Centralized reagent repositories, ... rapid access to validated reagents, especially during a time when these materials may not be available...for further investigation of this pathogen. The sharing and distribution of cell lines from repositories...community will continue having access to plasmids during the COVID-19 pandemic. Over 7,000 plasmids for ... Case study 3: Mouse strains Reagents developed during the SARS-CoV pandemic more than a decade ago are...et al., 2006). Now that a similar phenomena is appearing with regards to SARS-CoV-2, this mouse strain ...

... course the rapidly changing world of genome engineering via CRISPR.  Though the individual topics are...

...been able to progress so rapidly is scientific sharing. Already we’ve seen articles about COVID-19 on ...centralized databases and centers to support the sharing of materials and data associated with COVID-19 ... these resources and why they value scientific sharing as the scientific community strives to understand... motivations vary, everyone we spoke with sees sharing and collaboration as the best way that the scientific...are supporting their access to useful resources during this time. Facilitate collaborations within ...researcher studying COVID-19. “Open scientific sharing is important for creating research synergies and...iterating on methods for coronavirus research, sharing resources for COVID-19 research in their field ...

...determine functional titer with a colony formation titering assay or similar protocol. After functional titer...skewed the representation of your pooled library during the amplification process. It’s far better to verify...worked.  Ready, optimize, transduce!  If you’re ordering a pre-made library, you don’t need to worry about...optimize before you can transduce your cells.  Titering After generating your lentivirus, you’ll want ...specifically to allow scientists to perform functional titering and optimize transduction efficiency. Transduction...page from our book and put aside an aliquot for titering and optimizing before you freeze down your viral...and validation of CRISPR libraries. The Genome Engineering using CRISPR/Cas Systems Forum- Moderated by ...