...identified by various enzymes and sent to the proteasome for degradation. The tags we will discuss here...can yield intermediate protein abundance. Finally, some degron tags can do more than just degrade! Read ...will review the 4 most common types of degrons and some of the nuances to each one.   SMASh Small molecule-assisted...cleavage sequence. The tag is then shuttled to the proteasome due to the presence of the degron, and the protein...domain shuttles the entire tagged protein to the proteasome for degradation. In addition to having the coolest...protein is not amenable to being tagged, e.g. tagging some proteins can affect their function, the SMASh tag...system also requires less genetic engineering than some of the other systems reviewed here. The only required...

.... So… why do some people still use amp? Carb is expensive (relative to amp), and for some applications...During the propagation and/or transformation process, some bacteria will either never receive a copy of the...successfully used for plant and bacterial culture, but some types of bacteria, such as SHuffle cells, are already...colonies? Ampicillin Fair {% icon icon_set="fontawesome-5.14.0" name="Check" style="SOLID" height="16...Check icon" %} Just bacteria {% icon icon_set="fontawesome-5.14.0" name="Check" style="SOLID" height="16...Bacteria and eukaryotes (G418) {% icon icon_set="fontawesome-5.14.0" name="Check" style="SOLID" height="16...decorative" title="Check icon" %} {% icon icon_set="fontawesome-5.14.0" name="Check" style="SOLID" height="16...

... work and the rest of life is sometimes more of a struggle and sometimes less. When we are lucky, we are... make a change. #2 Thou shalt sometimes put work ahead of life Sometimes, work has to come first. That...can, pay someone to do the “jobs” that take you away from having your personal time. Someone else can ...list, and a to-don’t list Prioritize! Some things can wait and some things can’t. I kiss my kids good night... in the family to have to miss something – for example, say no to some of those birthday parties. Tell... can’t make it “all better” for you, I can offer some tips and tactics that have worked for my friends.... Tip: take turns going; that way you can have someone there clapping more often than not. You want your...

...in the pair comes from a topoisomerase I-linearized backbone. DNA topoisomerase I (depicted as a green ...covalently linked to topoisomerase. Vectors in these kits also often have the topoisomerase site inserted into...Topoisomerase based cloning (TOPO cloning) is a DNA cloning method that does not use restriction enzymes...technique specifically uses Vaccinia virus-isolated topoisomerase I as this enzyme recognizes the DNA sequence...cleaved 3′ DNA strand and a tyrosyl residue of topoisomerase I (1). If a 5′ hydroxyl group from a different...thus joining the two DNA strands and releasing topoisomerase (2).  These days commercially available TOPO...may want to add extra salt to your reaction: Topoisomerase I is released from the vector when the PCR product...

...tools as some kind of a meta/Frankenstein app? Or perhaps there is a clear winner, is there something that... sgRNA design is not possible without relying on some kind of pre-analysis. At the end of 2014, I began...the piñata, their role is to fill the prop with something of value. They are reviewers of content. On the...trust in the piñata builders - the reviewers. After some physical exertion on the part of the party goers...and its contents spill to the ground. There is something akin to a frenzy as the crowd tries to weigh and...what the reviewers so painstakingly assessed to something a bit more representative of the communities’ ...design tools The goal of this post is to provide some insight into available CRISPR software tools, what...

...I met some very accomplished scientists and some rising stars in the world of microbiology, some before...willing to introduce myself to someone new and ask for help. That was NOT something I would have done at my ...Mentor-Singleton relationship. Some of the variability was organic, as some people need less assistance ...conferences   We took some big steps after our initial pilot, and look to take some leaps. Binning Singletons...with me talking about the tools I could use, and some experimental hurdles I would encounter. This was...large meeting. They had the confidence to meet someone new, as they’d been introduced to several people...similar fields (ASM Microbe has eight tracks), something we didn’t have enough people to do in the pilot...

...companies (along with some research laboratories) have picked up the system for some of the uses outlined...human-like’ glycosylated proteins. Peroxisome studies Peroxisomes are small, membrane-bound organelles...phaffii is an excellent model for peroxisome studies since their peroxisomes will proliferate upon a media...name of beer and bread. The following table lists some of the biological commonalities and differences ...Facultative anaerobe Obligate aerobe Number of chromosomes 16 3 4 Preferred carbon source Glucose.... They are found in all eukaryotic cells and peroxisomes are where methanol metabolism occurs in methylotrophic...media change into methanol and peroxisomal autophagy can be induced by removing methanol from the media. ...

...which chromosome the transgene has integrated and make genetic crosses with balancer chromosomes to make...transgenic flies to study your favorite gene (YFG).  Sometimes, you want to use the Gal4/UAS system, but the ...To begin this process, you will first need to do some molecular cloning. Depending on whether you are ...like the gene to be incorporated into the genome? Some vectors incorporate into the genome randomly, while...that the transgenes can be inherited on the same chromosome. Targeted Insertion allows for direct comparisons...longer because you need to determine in which chromosome your transgene has integrated. This can be accomplished...accomplished with a few matings to balancer chromosome lines but is not necessary with targeted insertions...

...level of nucleosomes, loops, chromosomal domains, or how stretches of DNA on different chromosomes interact...when I recently attended the 2018 Chromosome Architecture and Chromosome Organization Keystone Symposium...posters I saw at the 2018 Keystone Chromosome Architecture and Chromosome Organization. These aren’t the ...chromatin architecture to every diagram portraying some aspect of eukaryotic DNA would become busy and potentially...linear” is a misnomer. The DNA packed into our chromosomes is very much 3-dimensional, as you can see by...Shapes a Single Nucleus from a Set of Mitotic Chromosomes. Samwer M, Schneider MWG, Hoefler R, Schmalhorst...

...Saccharomyces cerevisiae; however, we’ve also noted some features required for the replication of fission... host DNA or is independently replicated as an episome. The four main types of yeast plasmids are defined...and must be integrated directly into the host chromosome via homologous recombination. Yeast Replicating...Replicating Sequence (ARS) derived from the yeast chromosome. As the name suggests, these vectors can replicate...replicate independently of the yeast chromosome; however, they tend to be unstable and may be lost during...replicate as though they are small independent chromosomes and are thus typically found as a single copy... vectors are stable without integration. Yeast Episomal plasmids (YEp): These are most similar to bacterial...

...– just 1 out of 10,000 cells on average! Without some means of quickly determining which cells successfully...replicate the plasmid in addition to its own chromosomal DNA, costing additional resources to maintain...the two categories may be a bit of a gray area as some bacteriostatic reagents can kill bacteria when used...Concentration** Kanamycin aminoglycoside Binds 30S ribosomal subunit; causes mis-translation Bactericidal 50.../mL Spectinomycin aminoglycoside Binds 30S ribosomal subunit; interrupts protein synthesis Bactericidal...100 ug/mL Erythromycin macrolide Blocks 50S ribosomal subunit; inhibits aminoacyl translocation Bacteriostatic...100 ug/mL Tetracycline tetracyclin Binds 30S ribosomal subunit; inhibits protein synthesis (elongation...

...dinner saying something like, “I’ll be there in a few minutes, I just need to split some cells.” Inevitably...friends has a somewhat immediate deadline - even the best of friends get hangry at some point. My cells...by offering advice on areas where you might need some help. Today in our transferable skills guide: Time...larger payoffs later. Don’t be afraid to accomplish some quick but moderately useful tasks at the expense...splitting anecdote, I probably could have grabbed some food with friends and come back to the lab to split...the other hand, could have waited. Both tasks have some utility, but I lost all the utility (fun) of eating...I remember things better when I write them down. Some people also find it useful to write out their to-do...

...They were different people with some very different opinions, and some of them would acknowledge later... of lapsed communication. Making some assumptions about how something should be communicated is necessary...I first realized I might have some minor skills in conflict resolution after college, when I discovered...be. If people or teams can’t see eye to eye on something and a disagreement festers, it can lead to hurt...resolving--will make you a better scientist. And some amount of conflict is actually critical in the business...conflict, whether it’s a conflict between myself and someone else or between two other parties, the first step...Person A one thing and inadvertently told Person B something else.") The majority of conflicts I address in...

...style match for your needs can take some research.  It helps to spend some time thinking about what your goals...coaching plans that are financially accessible. Sometimes only a few hours of coaching time can make an ...know who have had a successful coaching experience? Some university career offices, postdoc offices or professional...vouch for them. Trust your gut – only work with someone you connect with. Talk to the coach about their...for clear information on costs. Can you suggest some coaches? Yes I can. Please note that Addgene does...LinkedIn and Facebook to see their online personas. Some have online coaching tools that can be very effective...Maybe you just need help getting through your PhD? Some coaches focus on graduate students. For example ...

...a vector backbone containing a topoisomerase I cut site, toposisomerase I, and a taq polymerase-generated...There are some cloning methods that make working with multiple inserts a breeze. Among them are some of the...consideration is: The number of inserts ...as some methods are more amenable to combining multiple ...polymerase-generated insert. When mixed, topoisomerase I provides both the endonuclease and ligase function while 5’ ...overhangs left by Taq perfectly anneal with the topoisomerase I generated overhangs in the backbone. Once ...Origene, and NEB® are all good places to look for some helpful kits! Efficiency: Wouldn’t it be nice if...successful result? While there are never any guarantees, some cloning techniques do tip the scales in your favor...

...by offering advice on areas where you might need some help. Today in our transferable skills guide: Negotiation...grad student, I thought that “negotiation” was something that I would mainly do over the price of a used...unprepared. Mainly, I thought of negotiation as something done not by people wearing a lab coat, but instead...transferable skill? Negotiation as a zero sum game Some people think about negotiation as a zero sum game... argument but felt bad afterwards). Or worse, sometimes there’s no agreement at all and everyone leaves...for this person’s request? Your colleague is if someone can feed their cells this weekend - why? Do they...plans? What is most important to each person? Someone has to be the first author but another author can...

... are aware of a problem or a need for something, usually someone else has realized this too. Begin with...by offering advice on areas where you might need some help. Today in our transferable skills guide: Collaborating... To get this and other projects like it going, someone (likely several people at Addgene) had to take ...accomplish a variety of goals. For example, I worked with some microscopy enthusiasts to start a microscopy seminar... about your ideas and don’t be afraid to make something concrete happen. You may have more willing collaborators...department, I personally felt that I had to overcome some hesitation to ‘approach strangers’ and learn how...companies and then convince the contacts to give me some time to talk or to at least read my emails. Then...

...by offering advice on areas where you might need some help. Today: Career Planning Resources. The key ... do now is diversify. Read on for an overview of some of the resources you can use to better prepare for...beneficial during my own graduate career – and in fact, some of the very things that were so critical to me landing... your CV or business card. Be warned, though – someone accepting your CV doesn’t necessarily mean that...elaborated on later. As for what job fairs to consider, some of the best options are the Naturejobs Career Expo...Biospace holds career events all over the country; sometimes several per month, giving you a plethora of opportunities... Post your CV and make it visible, which will sometimes result in a recruiter giving you a call. Many ...

...trainee possible? It’s not a mystery – there are some practices that can really help! Following best trainee...Typically, you will identify a ‘scientific mentor’: someone who provides career advice and offers direction...receive technical mentorship from a ‘bench mentor’. In some instances, the bench mentor is also the scientific...papers or protocols yet, don’t be scared to ask for some! You can email or ask in-person “Are there any papers...understand the purpose of, or a different way of doing something then you’ve done previously) you can bring those...especially when you are in a new place, learning something new. So how can you remember everything? Write...you have questions or need a little assistance at some point during the procedure. Be an active partner...

...vector remains episomal, meaning it stays separate from the genome. One option for episomal vectors is to...prokaryotic ribosome, while G418 (geneticin) is effective at inhibiting the eukaryotic ribosome. One resistance...will be lost from the population. A bacterial chromosome has a single origin of replication (ori), where...lines that express viral machinery. Alternatively, some slow-growing cells, particularly mammalian cells...but it won’t allow you to select for your vector. Some antibiotics block important cellular pathways shared...across kingdoms by targeting a shared aspect of ribosomal function. A puromycin resistance gene on your ...vector to survive while the auxotrophic cells die. Some auxotrophic selection markers can be used across...