...control copy number and stability, which influence the plasmid’s ability to establish and maintain itself ...Understanding the characteristics of natural plasmids and how they function in nature can inform us on...others have a broad host range and can replicate and stably maintain the genes they carry among organisms belonging... many different phylogenetic groups (Jain and Srivastava 2013).  Recombinant vectors that are made from...from broad host range plasmids are more stable, can be easily cloned in different bacteria, and can be ...transferred to various experimental hosts (Jain and Srivastava 2013). They are also easily-shareable tools within...utilize diverse host replication machinery. For instance, the ori typically contains iterons that serve...

...rotation students, joining a new lab at any career stage, learning a new technique while visiting a lab, ...technical mentorship from a ‘bench mentor’. In some instances, the bench mentor is also the scientific mentor...mentor…but usually the bench mentor is a staff scientist, postdoc, lab manager, technician, or a graduate...there any relevant papers you can read prior to the start of your training? These could serve two purposes...and other educational resources to help you get started!) Are there protocols you can familiarize yourself...materials (maybe a step in the protocol you don’t understand the purpose of, or a different way of doing something...your bench mentor if they have time to answer outstanding questions you have, well before experimental ...

... its unbound state contains a fluorophore and a quencher in close proximity. For instance, the fluorophore...was less stable due its RNA nature, it paved the way to new biosensor possibilities. For instance, further...years ago, two independent groups, led by Jack Szostak and Larry Gold, developed methods for selecting... from experts on FRET techniques. By combining standard donor/acceptor FRET know-how with aptamers, aptabeacons...aptamer system. Knowing that GFP’s  structure stabilizes a 4-hydroxybenzylidene imidazolinone (HBI)-like...Spinach eventually evolved into Spinach2, a more stable and brighter version of the aptamer, but it still... of SELEX and FACS, Broccoli is inherently more stable in cells. The new aptamer also has a higher melting...

...regardless of visa or fellowship status, in addition to certain staff scientists and those in technical...$47,476 per year, under updates to the Fair Labor Standards Act (FLSA). This is having a major effect on ...students are exempted from the law. However postdocs, staff scientists and technicians in primarily bench-science...$43,692 to $50,000, an increase of 14.4%. In 2015, Stanford raised salary levels to $50,000, and as of October... The University of Notre Dame sets no minimum, stating that while they recommend NRSA levels, postdocs...not to be tracked. University of Buffalo and Ohio State both have minima of $30,000, and raising salaries...addition, both Brown and Brandeis universities have stated that they consider postdocs paid on stipends to...

...schools and was started in 2005 in Kansas by Bobby Henderson, a physics graduate from Oregon State University...through synaptic connections (Shepherd, 2004). Understanding the structure and function of these neural circuits...researchers often want to do at neuronal synapses for instance. Also these tags need to be fused to scaffold ...scaffold proteins in order to be expressed stably in neurons. As compared to epitopes tags, FPs are intrinsically...circuits and dynamics. FPs are generally bright, stable and well tolerated by cells. They can be used for...The issue with these FPs is that they are less photostable than GFP. In addition, antibody-based amplification...useful for multicolor imaging experiments than standard FPs. The Looger Lab was thus able to easily perform...

...is also an avid long distance runner, gardner, and cook.      Lindsey M. Costantini received her PhD in...by guest bloggers Erik L. Snapp and Lindsey M. Costantini. "You underestimate the power of the Dark Side...non-cytosolic protein functions properly? We suggest starting by using the newly published oxidative optimized...used in various combinations. For example, with standard fluorescent microscope filter sets, users can ...when utilizing an optimized moxFP as opposed to standard FPs. The increase in signal is not due to a brighter...systems should strongly consider using moxFPs over standard FPs for the reasons listed above. In addition,...susceptible to the oligomerizing effects of many standard FPs. The moxFPs are now available through Addgene...

...about Synthetic Biology. Can you tell me where to start?” is a question more and more students are asking...and joy, rather than a perfect lesson plan or outstanding test scores.  As a result, Kuldell launched the... high school teachers to boost interest and understanding around the engineering of biology. Participating... principles to biological systems. Today, what started in Cambridge with a handful of local high school...BioBuilder curriculum is now taught in almost every state in the U.S. and in more than 30 countries around...recognized platform in March to exhibit projects at any stage of completion, from pitch to prototype. Last year...breathalyzer to pesticide breakdown, production of fire-resistant housing insulation, and cleaning up salt runoff...

...method requires constant turbidity of the culture, so bacteria must be grown in a turbidostat. Only mutate...spectinomycin resistant phenotype in bacteria. While there are many known spectinomycin resistance mutations...only uncovered novel resistance genotypes, but also provide a better understanding of the key residues ...of wild-type E. coli, while only increasing the standard mutation rate seen during DNA replication by 120... than the original nCas9, while increasing the standard mutation rate by only ~2-fold over nCas9 levels...allow researchers to trace the progeny of a single starting cell. Additionally, multiplexing of guides allows... feature which they used to generate bacteria resistant to both spectinomycin and streptomycin, but could...

...grateful, I started setting aside 3-5 minutes to sit quietly at my bench before the start of any experiment... you’re much more likely to make a mistake. When you make a mistake, you’re more likely to be stressed...experiment failing because of a small, avoidable mistake. Or worse, when you're trapped in a pattern of ...same goes for your workflow. Next time you make a mistake, instead of silently vowing never to do that again...workflow, or your physical setup - to prevent the mistake, just like you would change variables to fix a ...a failed experiment.  For example, I used to constantly underestimate time needed to prep for an experiment...experiment. What fixed it was not trying even harder to start earlier, but simply moving all my prep work to the...

... an Assistant Professor at Brigham and Women’s Hospital. Biochemists often struggle to understand how ...molecular distances (see Table). However, proper control experiments are needed to establish the dynamic...acceptor (D/A) distance at which 50% FRET occurs. b Range corresponds to calculated D/A distances for observed... FRET can be used to derive a precise physical distance between the donor/acceptor fluorophores (Stryer...labeling. Fluorescent proteins (FPs) are the gold standard for genetically-encoded FRET donors (Fig. 1). ...especially since the potential rewards could be substantial: Plasmid tRNA Synthetase Expression Host ...reviewed in [Yan & Bruchez, 2015]). To get you started, two robust orthogonal labeling strategies are ...

...Justin Ng and Alyssa Cecchetelli Science is a constantly evolving and demanding field, requiring a variety...of biological research that has defined our understanding of biological processes and developments. Model...used as a model organism for over 60 years with a status as the most studied prokaryotic organism. With ...growth rate, small size, and low cost, it’s understandable why E. coli is the most popular bacterial organism...settings. One of its most attractive features is its stability and easily obtainable growing conditions. As a...thrive with or without oxygen. Additionally, its status as a Biosafety Level 1 microbe makes it a perfect... biofilms, and much more. Because of it’s BSL-1 status, E. coli can be used as a model to relate these...

...chloramphenicol-resistant strains, we have also included versions with gentamicin-resistance (amplified from...disrupt expression of a gene with an antibiotic resistance cassette. This leaves a “scar” in the chromosome... in the creation of scarless mutations, both in stand-alone systems (Jiang et al., 2013), and those which...be accomplished by selecting for an antibiotic resistance marker from the vector (abR, Figure 1). The resolution...This vector contains sacB, an chloramphenicol resistance cassette, and the R6K origin of replication to...replicated in the recipient, the only antibiotic resistant colonies isolated are those where the allelic ...conjugate that well. This is because in these circumstances, the conjugation can be so inefficient, that...

...between studies is substantial (e.g, the poor performance of mRuby2 is detected by Constantini et al (2015) ...selecting a bright fluorescent protein and a photostable fluorescent protein. In the third post of this...modules that fail to homodimerize in cells. To understand how well fluorescent proteins approach this ideal...dimerization tendency, as is present in EGFP. Constantini et al, (2012) developed a cell-based assay that...tagRFP does not act as a true monomer in cells (Constantini, 2012), whereas it was previously concluded to...chosen FP in your cell type of interest before starting your experiments.  Selecting a non-oligomerizing...CytERM with mTurquoise2 and dTomato, which are established monomeric and dimerizing FPs respectively, are...

...scicomm bandstands: cognitive load.  Cognitive load The Wikipedia definition of cognitive load states: “cognitive...mental thought and effort your audience needs to understand what you’re communicating. While I’m sure that...of knowledge new to mankind. And you probably understand how much faster it would be for an expert in ... are asking your audience to take on, just to understand you at the most basic of levels.  Concepts This...lot of science communication is helping people understand new concepts. You hopefully are aware that this...foundational concepts (that they often don't!) to understand us, or being able to connect one concept to another...for your audience. I ask myself that question constantly (and so should you!)  Amount of information and...

...organisms can’t use. Other plasmids offer resistance to toxic substances like heavy metals or pesticides, enabling... Blog Plasmids 101: Antibiotic Resistance Genes Antibiotic Resistance: An Old Solution but a New Problem...natural plasmids. Created with BioRender.com. Resistance (R) plasmids: microbial defense Genes allowing...its host. For example, cells with a penicillin resistance plasmid can grow in spite of a hostile Penicillium...fungus trying to kill them off. While antibiotic resistance-mediated selection is indispensable for cloning...cloning work, antibiotics and plasmid-encoded resistance to them are the tools of a millennia-old microbial...like colicin typically also carry the necessary resistance genes to help cells survive their own secretions...

... for antibody expression. If starting from a protein sample (for instance, affinity purified antibodies...then be tested for antigen binding. If starting from an established hybridoma line, the antibody can be ...from a plasmid or from an integrated sequence in a stable cell line. The genes encode the heavy chain and...traditional hybridomas.  First, the long-term stability, consistency between batches, and molecular definition...and sequences for antibodies are not shared. For instance, through open plasmid sharing, the CRISPR community...antibodies, modify antibody function, improve antibody stability, and design tools that we haven’t even imagined...10.1038/521274a Begley CG, Ellis LM (2012) Raise standards for preclinical cancer research. Nature 483:531...

...combined in one step. As with standard IF, you will need to start with a fixed sample. Depending on... antibodies. The general process is similar to standard immunofluorescence, but with some extra considerations...photobleaching or by stripping out the antibodies, then start over with the next target: add the new primary, ... will need to include a marker like the Hoechst stain for the nuclei. (See Brewer et al, 2022 for a sample...Abcam's sample protocol is a good place to get started.  One important consideration when deciding whether...magenta), anti-AnkyrinG (green), and Hoechst nuclear stain (cyan). Panels C1-C3 show magnified detail of the..., many of the same considerations apply as for standard IF. However, there are a few specific considerations...

...time, plasmid prepping is a breeze. You get your stab from Addgene, streak for single colonies, sub-culture... genotype, as these mutations improve plasmid stability and yield. If a plasmid is prone to recombination...plasmids with inverted terminal repeats. We use NEB Stable to transform and prep high quality DNA that contains...recombination, but are also endA+. endA encodes a thermostable periplasmic endonuclease that can sometimes ...of common E. coli strains. Increase the volume Starting with a larger culture volume can improve plasmid...performing filter based plasmid prepping. Be sure to stay within the recommended range of the filter or you...growth conditions. For many high copy plasmids, standard LB broth and antibiotic concentrations will work...

...forget to start it, the samples can actually migrate into the buffer — so it’s better to start over if ...consider using an unstained protein ladder, which will run a bit more true to size than a stained ladder. Unstained...transfer worked with Ponceau S staining or other reversible protein staining immediately after your transfer... Check transfer with Ponceau red stain or other reversible stain* Roll out all of the bubbles in...dilutions of your sample or protein of interest to understand the dynamic or working range of a given reagent... runs to optimize or troubleshoot.   Before you start… Mark your membrane! Membranes look the same from... amounts of protein, make sure your reversible staining method of choice is appropriately sensitive. If...

...of preference, and it will be stable for years. Plasmid DNA can be stable at 4°C or even room temperature...answer: yes. This last step will influence the stability of your sample over time and determine which experiments...effectively use the DNA. pH and DNases affect DNA stability Figure 1: So many choices! What to do? ... The main factors that can affect DNA stability in solution are pH and DNase contamination.  Extreme... favors the breakage of the hydrogen bonds, destabilizing the molecule and favoring the separation of ... be able to store your sample without having substantial degradation and which experiments you can use... buffer is the best buffer for preserving the stability of your preparation for a long time. Tris buffer...