...tetracycline resistance gene and the lambda red repressor cI857.  Once introduced, the prophage is stable and ...dsDNA-dependent exonuclease.  Exo will degrade linear dsDNA starting from the 5’ end and generate 2 possible products...replace a gene of interest with an antibiotic resistance cassette.   Substrate DNA Design and Generation... selectable DNA fragments, such as antibiotic resistance genes, as well as non-selectable DNA fragments...an easy fix, but can only be used in limited circumstances. An alternative approach is to flank the desired...prophage A number of E. coli strains exist that stably express lambda red recombineering genes due to ...℃.  Mini-λ A hybrid between using a plasmid and stable integration of a defective prophage is to use mini-λ...

...cells with a membrane possess an electrical state. This state, known as the resting potential (Vmem), can... plasma membrane specificity. Stable Step Function Opsin - A stabilized step function opsin (SSFO) for...collection of bi-stable anion optogenetic channels which can be toggled between open and closed states using different... junction proteins cx43 GJB1 GJB2 Bioelectric states can alter cell types and patterning There is mounting... or cancerous it is (Levin et al., 2017). These states can even be necessary and sufficient for certain...visualize or perturb these slower-changing bioelectric states in living cells. Since bioelectrical signals are...indicators (GEVI) are ideal tools to track bioelectric states (Matthew et al., 2018). These allow for not only...

... side of the grant to be established. Take time to read and fully understand the RFA. The RFA contains.... Now is the time to make bold statements. While some of the statements may be slightly far reaching, ...italic, or underline font formats helps these statements stand out from the rest of the text. Specific aims...always helpful to remember that if a PI asks for assistance in the grant application process, she has faith...opportunity to summarize the limitations of current state of the art technologies. Therefore, take time to...of current methodologies. This section sets the stage for why your funded proposal will address these ...preliminary data suggests the feasibility of the statement, use it to confirm that your solution is the best...

...select your state. State Legislatures: To find the website to contact representatives at the state level you...author do not represent a formal stance taken by Addgene or its staff. In Washington, D.C. as well as ...author do not represent a formal stance taken by Addgene or its staff. Many thanks to our guest blogger...for seeking knowledge. The process begins with a testable hypothesis - an educated guess about how some ...march in Washington, D.C., the seat of the United States government? Politics and science are intrinsically...literacy as I believe people that can see and understand data themselves can come to conclusions themselves... you can click on your state in this map. Finally, the march is meant to inspire scientists to be more...

...increases plasmid stability recBCD Exonuclease V activity abolished Increased plasmid stability, reduced recombination...of 30S ribosome Confers resistance to streptomycin Tn10   Confers resistance to tetracycline Additionally...optimal promoter for your gene of interest, painstakingly cloned into the perfect empty backbone, made...high-throughput cloning, routine cloning, cloning unstable DNA, preparing unmethylated DNA, and more. Many...dnaJ Mutation in a chaperonin gene Increases the stability of certain expressed proteins endA, endA1 Endonuclease...uptake, iron uptake receptor mutation. T1/T5 Phage resistance gal Mutation in galactose metabolism pathway...only gyrA, gyrA96 DNA gyrase mutation Confers resistance to nalidixic acid  hsdRMS hsdR(rk-, mk+) Unmethylated...

...) the courage to make mistakes and 2) the integrity to recognize these mistakes quickly. However, many...same atmospheric physics to understand exoplanets that we use to understand the Earth! Denver, CO, USA ...we, scientists and non-scientists alike, need to stay active and involved if we wish to make positive ...role of science in our democracies will require constant vigilance and actions beyond marches, as well ...Institute, most women of reproductive age live in a state with at least two abortion restrictions that conflict...not only believe in the separation of church and state, but also the separation of fact and fiction. Our...depends on it. Deanna McLafferty is an editorial assistant and activist. Washington, DC, USA 38.9072°N 77.0369...

...in an open inactive state, a closed active state, and an artificially active state due to closure by FP...long distance runner, gardner, and cook.   References 1. Snapp, E.L., et al., Formation of stacked ER cisternae...you are using a tag with a serious drawback. The standard EGFP plasmid that used to be sold by Clontech ...its molecular affinity, termed the dissociation constant or Kd, and its concentration. The smaller the ...membranes together and can wrap the membranes into stacked structures including organized smooth endoplasmic... to a dimerizing FP has potential to form large stable polymers of dimers. 3) FRET biosensors that contain...of tubules can be grossly distorted into dense stacked membranes termed Organized Smooth Endoplasmic Reticulum...

... worked from Standard Operating Procedures (SOP’s) and every item I tested was constantly tracked with...touch with my colleagues and have misunderstandings.  Working long distance, you need to be very self-motivated...people from another background or culture could understand. I did, however, find it very hard to speak with...to be in the lab doing experiments  without the constant fear of failure because I knew the tests worked...the different tests and became efficient, I then started to become bored because I was not being mentally...mentally challenged. For these and other reasons, I started  looking for other career options after 6 months...redundancies can be made within the company; new staff hired in anticipation of the contract suddenly have...

...always a good idea to start close to your comfort zone and slowly expand from there. Start small: Collaboration...implementation, and now must focus on managing the established partnership. If you’re willing to do similar ...my graduate studies at Karolinska Institutet I started working with other groups. First within my department...example, I worked with some microscopy enthusiasts to start a microscopy seminar to connect the imaging community... scientific collaboration - show initiative and start organizing meetings and take minutes. Get to know... little in common with you is much harder than starting a collaboration with a colleague. However, reaching...discussing with strangers stopped feeling weird and started to be fun. I just had to remember to approach the...

... an antibiotic resistance gene to allow for antibiotic selection, but these resistance systems don’t always... develop resistance to selection toxins. Auxotrophic selection is useful for producing stable mammalian...of bacterial transformation is transfection. In stable transfection, DNA from the plasmid is incorporated...transfection, the vector remains episomal, meaning it stays separate from the genome. One option for episomal...Mammalian cells At least one of the following: to stably transfect the vector a viral ori in a compatible...shared aspect of ribosomal function. A puromycin resistance gene on your vector can protect both plasmid-...also block protein synthesis, and the neomycin resistance gene protects against any of them. But different...

... job and job requirements. It’s okay to start from a standard version, but take some time to make it fit...your application, but it can certainly help you stand out.  In some fields, a cover letter is vital, particularly...a good idea to talk to people in the field to understand what the norms are. What is a cover letter for... to master ddPCR.  Finally, it’s a good idea to state why you’re applying to the specific company or lab...struggling to think of what to put in your cover letter, start by looking at the requirements in the job description...rationale for designing it the way you did, and state that it would help you manage a large number of ...yourself and your background. It’s a great place to state why you’re applying to the job, company, and/or ...

...high school to college to graduate school, standing at the start of a science policy career was much more...for science policy folks is on Twitter- start an account and start engaging with the #scipol hashtag. Often....  Who does science policy? I’ve intentionally started with the ‘who’ question because the science policy... from your alumni community is a great place to start. What is science policy? THE IMPOSSIBLE QUESTION...branch or in Congress, or can also happen at the state and local level. The big secret about science policy...tenured professors. There’s never a bad time to start your science policy journey.  In fact, working in...your career through engagement with your local or state governments. Most communities have opportunities...

...biology, innovative new imaging approaches are constantly being developed which combine computational processing...this. This recent push gives users the chance to understand the "black box" provided by the manufacturers.... 1: Open source hardware builds   Since I started my PhD, I have been convinced that most microscopy...creativity and feedback from the community to create state-of-the-art devices for research. There are a number...support the community in developing better, more sustainable, easily sharable products. Open-Source Microscopy...time-lapse images, quantitatively evaluating antibody staining, or counting cells are now routine, open-source... process framework FIJI are just three of the constantly growing number of "community-driven" software...

...Challenge #2: Technical obstacles to plant synthetic biology In addition to public resistance, an overall limited...limited understanding of plant biology is a significant technical obstacle to plant synthetic biology. We.... Read their previous blog post about how they started their company here. The first transgenic plant ...technology. Monoculture and chemical spraying, mainstays of modern agriculture, are now synonymous with...imagine any use for GM technology other than to sustain those techniques. We feel strongly that the scientific... We cannot build what we do not understand, and the complicated and unique metabolic processes of plants...project in a field with 30 years of basic research standing behind it. Our color changing flower involves ...

...bind DFHBI, is highly unstable in vivo due to poor folding and thermal instability at 37°C (Ouellet et ...to the instability of Spinach, and were able to develop an aptamer that was more thermally stable, exhibited... not photostable, and were presented in the study to support the uniqueness of the photostability of the...) and requires a tRNA scaffold to improve its stability in vivo. However, Spinach has proven useful in... RNA. Spinach-based riboswitches can be used as stable metabolite sensors for imaging in live cells. By... fluorescence. In the search for something more stable for quantitative imaging, Ming Hammond’s group ...lower dependency on magnesium and higher thermal stability, making it a potentially more useful tool for ...

...imaging is an easy way to understand what is going on in the body at steady state or under physio-pathological...adult organisms, where tissues structures are more stable and local movements predominate (e.g. a basal progenitor...progenitor cell migrating to the epithelium). At this stage, only cells from the immune system show wide scale...information regarding immune cell mobility than the static microscopy images of chemically coloured lymphoid...immunologists the ability to count cells and statistically analyze cell movements. By using cells labelled...two-photon microscopy allowed scientists to better understand T cell initiation in the adaptive immune response...uncovered host-pathogen interactions leading to the understanding of effector function in infected tissues. Before...

...for you, don’t wait. Start communicating now. These 9 strategies will get you started:  Subscribe to Career...Take the DIY approach and start a blog. Stellar talents like Ed Yong jumpstarted their science writing career...Twitter account. Check out the hashtag #scicomm and start following science communication societies like @...local pub at a Pint of Science event. Or get on stage for a Science Slam – a sort of competitive TED talk..., set up a blog using a site like WordPress and start writing. Grow your audience by advertising your ...are open to guest contributions. If you decide to start your own blog, check out these two excellent articles... is the path you want to follow, check out the distance learning courses at the National Council for the...

... The STAR interview method is a popular way to answer behavioral interview questions. STAR stands for ... describe all four parts of a STAR answer once the interview has started. Instead, I preferred to prepare...was my experience not long ago. I just recently started working in the pharmaceuticals industry after earning...several individual and/or small groups, usually starting with the hiring manager. These interviews lasted...Story telling shares many of the same elements as a STAR answer: a setting, a conflict, and a resolution....which is something not inherently captured by the STAR method. I like to think of the theme of my interview...resumes are like cliff notes, so I use these as a starting place for drafting my stories. In total, I prepare...

...antibody to stain a target protein in mouse tissues, causes a high degree of background staining. This occurs...secondary antibody) approach?  Staining for multiple targets When staining multiple targets indirectly, ...for a four-antibody panel, staining in parallel    In short, when staining multiple targets, look for...validated for your application, of course!) When the stars are aligned and the lab gods are smiling down at...the tissue sample. There are species-on-species staining protocols that use antibody fragments to block...endogenous immunoglobulins and reduce the background staining but they do not always eliminate the issue (Lu...better to choose primary antibodies from more distantly related species and/or use narrowly specific secondary...

...to gain support for CINSO. For instance, I tweeted to the Indiana State Museum and others when I was trying...engaging the public to promote an awareness and understanding of science. Like most science PhD programs, ...often called “nontraditional,” helped prepare me to start CINSO. Here, I share some of the tips I learned ...up and moved on to the next thing. If I made a mistake, I evaluated what lessons I had learned – call ...the process. As I mentioned, my graduate office staff was amazingly supportive. I talked to them about... had to take advantage of it. Blog and tweet I started my blog, Born to Science, as a graduate student...experience with my blog helped immensely when CINSO started brainstorming the resources we wanted to provide...