... While antibiotic resistance-mediated selection is indispensable for cloning work, antibiotics and plasmid-encoded...the Addgene Blog Plasmids 101: Antibiotic Resistance Genes Antibiotic Resistance: An Old Solution but...now used routinely in biotechnology to transform plant cells. Degradative plasmids: microbial hardiness...environments — like by living where no one else wants to. Some organisms manage this with plasmids that...created as whole-genome sequencing becomes more relevant (Garcillán-Barcia et al., 2023). In this post,... antibiotic...

...companies that produce GMO food plants. When a press piece ran on the detector plants, we expected to see that... lab focused on product development: building a plant that could sense and respond to novel substances...Facebook comment.  In our day job, we worked on plants engineered to detect explosives and rapidly indicate... a beautiful GMO, one that people would actually want to have. Once we had the idea, we knew it was a ...It has also allowed us to really analyze what we want out of our careers: to build something new and beautiful...we are building this company, and that’s what we want it to do. It’s exhilarating to have that sort of...have great partners in this accelerator program who want us to succeed. We’re excited to see what the next...

...third post will describe how you can make your own mutant flies. Find Drosophila Resources at Addgene The...combs. To make genetic crosses properly, it is important to isolate females from males while they are still... after hatching into their adult phase. It is important to clear out the parent crosses from the container...method for maintaining stable lines of genetic mutants, including in situations where maintaining heterozygosity...feature of balancer chromosomes is that they produce dominant phenotypes visible on the flies. For example, ...feature when setting up genetic crosses. More importantly, this knowledge will stop you from embarking ... of Fly Pushing by Ralph J. Greenspan. It’s a fantastic book for newbies on how to work with flies. ...

... and this nuclease-null or “dead” Cas9 (dCas9) variant can then be fused to effector domains such as transcriptional...limitations to a system that relies on two distinct Cas9 variants—nuclease-competent or nuclease-null—in order to...with both the nuclease-positive and nuclease-null variants of Cas9 along with the necessary gRNAs, but it...control which gRNA becomes complexed with which variant of Cas9. To circumvent this problem, previous studies...suffers from several limitations. Perhaps most importantly, most Cas9 orthologs are less well characterized...target site. Benefits of truncated gRNAs Importantly, the use of truncated guides has largely been...graduate student at the University of California, Santa Cruz, where her research is focused on engineering...

...strains, a change in media may be advantageous. Reduce the antibiotic concentration Low copy plasmids produce...additional steps are needed to optimize yield. You might want to include an extra wash step or use a different...suggest retransforming the plasmid into a strain meant for DNA purification. It sounds like extra work,...many high copy plasmids, standard LB broth and antibiotic concentrations will work just fine; however, ... produce fewer transcripts of their antibiotic resistance genes and can therefore be cultured in media...media containing half the normally recommended antibiotic concentration. Add supplements to the media You... Resources on Addgene.org Find Info on How to Quantify Your DNA Learn to Do a Bacterial Transformation...

...differently and you may want to select one (or both) depending on the information you want to verify. We will...Organism box. This small piece of information can significantly reduce your wait time for blastn, blastp, and...expected ORFs, mutations or truncations. A primary advantage of blastx is that you do not have to decide on...using blastx at Addgene, we use the default “Non-redundant protein sequences (nr)” database as it contains...recommend using blastp over blastx. The primary advantage is time savings but an added benefit is that blastp...the default scoring matrix BLOSUM62, but you may want to check the description of the other matrices to... to see if another would be more advantageous for your search. Timesaving Tip #3: Note that protein databases...

...for science, and numerous other reasons. It is important to remember that we, scientists and non-scientists...role of science in our democracies will require constant vigilance and actions beyond marches, as well ...and calling for billions of dollars in cuts to important research funding.  So when I saw that there was...depends on it. Deanna McLafferty is an editorial assistant and activist. Washington, DC, USA 38.9072°N 77.0369...scientist.  I care about my work because it's important to our health.  Most people won't argue with that...been so well-publicized, we might encounter hostile anti-science activists. However, I must say that I was...I’ve worked tirelessly to get the publications, grant funding, and teaching experience that it takes to...

...CRISPR in plants, to learn techniques for detecting genome edits, to discover why you might want to use ... sent Outreach Scientists to Colorado, Berlin, Atlanta, Ottawa, Norwich, and Hannover, we raised money...popular blog post this month shares incredibly important information for anyone considering a postdoc. ... through your PhDs. Of course there were many fantastic posts in October. Click on the links below to ...Advice Part 2: Building Community Engineering the Plant Genome Using CRISPR/Cas9 Healthcare Consulting: ...Door to the Business of Life Sciences 6 Tips for Grant Writing 5 Reasons to Use Reddit for Science Communication...

...gut, and other organs to the CNS, are also an important target for gene delivery, especially for the study...Targeted Evolution. In this technique, a pool of mutant AAV capsid (cap) expressing plasmids is generated...infection and screening by PCR enrich for capsid variants with increased ability to infect the cell type...AAV.PHP.eB and AAV.PHP.S to their parental vectors. To quantify this comparison, capsids were packed with a GFP...out tables 1 and 2 below for a summary of the quantified transduction efficiency of AAV-PHP.eB and AAV-PHP.S... BP, et al. Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain...3293995. Additional Resources on the Addgene Blog Important considerations when using AAVs Learn more about...

...in close proximity and present in large enough quantities (and the correct stoichiometric ratios) to generate...generate useable data. What’s more, measuring and quantifying FRET is often easier said than done. Fortunately...overexpression of fusion proteins it requires can lead to aberrant subcellular localization of the studied PIPs. ...protein construction, cellular expression, and quantification (Kerppola, 2006, 2013). These are described... supraphysiological expression levels. BiFC quantification This is where BiFC outshines FRET — BiFC experiments...BiFC experiments. The ideal control is a mutated variant of one or both of your PIPs in which the protein-interaction...requires that you empirically test a number of mutants to identify those that inhibit BiFC in your cellular...

... as well as affinity purification with an anti-Cas9 antibody. These different tag systems and affinity...Using similar affinity purification with an anti-Cas9 antibody, the Li lab found a microRNA (miR483) that...transcription factor IRF-1. Immunoprecipitation, using an antibody targeting the 3xFLAG tag, allowed these researchers...this form, the CRISPR complex, consisting of a recombinant dCas9 protein and synthetic gRNA, is incubated...this technique, researchers have begun to make important discoveries in various biological fields. In 2015...chromatin by in vitro enCh IP technology using recombinant CRISPR ribonucleoproteins." Genes to Cells 21.4...

...genomic deletions. What are the advantages of Cas9 RNPs? There are many advantages to using RNPs for CRISPR ... Delivering Cas9-gRNA ribonucleoproteins  An advantage of performing CRISPR experiments using RNPs is... can be used to deliver a Cas9-gRNA RNP. This advantage has allowed scientists to perform in vitro and...et al. 2018). This was accomplished using a Cas9 mutant (Cas9M1C/C80S) that harbors two surface exposed...integration in crops, delivery of Cas9-gRNA RNPs to plants, as an alternative to plasmid based techniques,... account with these types of experiments. One important consideration is that the Cas9-gRNA RNP will be...it is likely that the use of RNPs will play an important role in its continued advancement. Andrew Hempstead...

...from a sample. Chemical fluorophores can be substantially brighter and more photostable than fluorescent...reverting” to small molecule dyes seems daunting—no one wants to spend their days microinjecting fluorescent conjugates...systems consist of a genetically encoded enzyme variant tag that reacts specifically and irreversibly with... lipoic acid ligase, biotin ligase, phosphopantetheinyltransferase) – These enzymes catalyze the covalent...Fluorogen activating proteins (FAPs) – These modified antibody fragments bind and enhance small molecule fluorogens...excess dye from the sample, which is particularly important for samples where washing is difficult, such as...Nevertheless, these fluorophores were designed as antibody and oligonucleotide labels for fixed-cell imaging...

...bNLS and NES sequences. Disadvantages of KTRs KTRs also have some disadvantages when compared to FRET reporters...least one site. Phosphorylation is particularly important for regulating signal transduction and measuring...proxy measurement of kinase activity. The key advantage of KTRs is that they are easy to create and simple...fluorescence serves as a proxy for kinase activity.  Advantages of KTRs When compared to FRET reporters for measuring... measuring kinase activity, KTRs have two key advantages. First, they are easy to multiplex. Their design...together. Second, KTRs are easy to design. The key determinants of KTR function are the positions of the phosphorylatable...

...Nature Communications Find the Cas12b plasmids  Plant base editing with a Cas9-adenosine deaminase fusion...fusion Caixia Gao’s lab has developed a new plant base editor from an evolved tRNA adenosine deaminase ... be used for A•T to G•C edits in protoplasts or plants of rice and wheat. Read the paper in Genome Biology...bacteria from various environments. It has the advantage of stable genomic integration and is easily transferred...three different Cas12a inhibitors. Some of these anti-Cas12a proteins have broad inhibitory effects on...human cells. Read the paper in Science Find the anti-CRISPR expression plasmids CRISPR-based method...recombinases used to excise markers. It has the advantage that CRISPR efficiency can be controlled by manipulating...

...in technical support including tips, advantages, and disadvantages of this overall rewarding role. Learn...high-impact journals, and the requirement for a high grant success rate. At this point, candidates are highly...the progress of the project. After that rather unpleasant experience I thought: I could do this job so ...additional data regarding the specificity of this antibody?” You will also help troubleshoot products, give...involved in such a role. In addition, it can be advantageous to speak multiple languages - that makes communication...possibly this same reason, it can be one of the more constant and safe corners in a company, less exposed to... in a Western Blot protocol and then blame the antibody when no signals are obtained. Or someone may heat...

...reporter can be measured and quantified to determine the presence and quantity of DNA, RNA or a mutation ..., scientists have utilized CRISPR-Cas9 protein variants, Cas13, and Cas12a, to develop simple, portable...fluorescent RNA by the “activated” Cas13 produces a quantifiable signal that indicates the presence of your targeted... pre-amplification step allowing for greater quantitation without compromising sensitivity. To detect... channel as the Cas13 reporter), they could significantly amplify the detection signal for a single target...CRISPR Trans Reporter Cas12a (Cpf1) is a CRISPR Cas variant that can also cleave dsDNA similarly to Cas9 (Zetsche...degraded at a rate of ~1,250 cuts per second. A quantifiable fluorescent signal designates the presence of...

... everyone who was doing the kind of work my lab wanted to do already seemed to know each other and what...and new friends while attending ASM Microbe in Atlanta, GA, in 2018. A lot of my experiences rang true...similar situations to themselves, allowed the participants to feel more comfortable at a large meeting....They really liked us.  The testimonials from participants in Binning Singletons at ASM Microbe in 2019...people to do in the pilot. We also found that all participants would benefit from more structure with regard...assistance than others, but we did have some participants feel like their mentors weren’t as invested ...expectations have been established for future participants regarding the activities and time mentors spend...

...however, in some cases it would be possible to grant those wanting to understand and/or attempt to reproduce...only ever a small fraction of all data directly relevant to a particular experiment, series of experiments...tool like an ELN. It is nevertheless critically important to be able to conveniently reference specific ...should have integrations with a broad range of relevant generic and science-specific tools. In addition...with a particular experiment or project thus significantly enhances the ability of others to subsequently...resources remain live. This means that someone who wants to query and attempt to reproduce an experiment ...Massachusetts, and feels privileged to be part of the vibrant life sciences research and commercialization community...

...not function properly in E. coli BL21(DE3) as transformants failed to grow after the transformation of pTargetF...make other edits. Once you have all the edits you want, cure pCas9 by growing the cells at 37 °C.     ... This would result in the lack of recovered transformants. Our speculation motivated us to update the ...the newly generated pEcCas/pEcgRNA system.   Advantages of the pEcCas/pEcgRNA system Compared with the...conclusion, the MUCICAT system has the following advantages: Fast and site-specific multicopy chromosomal...Sciences (CAS) Center for Excellence in Molecular Plant Sciences in 2019. She joined Sichuan Normal University...Lab of the CAS Center for Excellence in Molecular Plant Sciences since December 2020. His research focuses...