...number during the transition to stationary phase, Segall-Shapiro et al. placed a trans-acting RepA protein... the plasmid by altering IPTG concentrations. Higher IPTG concentration leads to higher RepA expression... the team made use of stabilized promoters. In contrast to unregulated promoters that are always on, stabilized...because TALEs can be programmed to bind tightly to arbitrary DNA sequences and have been shown to achieve ~...high plasmid copy number. When they increased concentrations of IPTG, a constitutive promoter upstream of...optimization of gene dosage in E. coli using DIAL strains." Journal of biological engineering 5.1 (2011):...): 10. PubMed PMID: 21787416. PubMed Central PMCID: PMC3163176. Resources on the Addgene Blog Read the...

...and IREB2, which post-transcriptionally regulate iron metabolism pathway transcripts. By using RaPID, Ramanathan... include both scrambled RNA motifs and cells transfected with BirA* protein alone (i.e. no RNA bait). ...experiments. These background proteins were then subtracted from Ramanathan et al.’s RaPID datasets. Applications...changes in RC2H1 protein expression and post-transcriptional regulation of SM1v1-containing RNAs could play...often trick host proteins into binding their untranslated regions (UTRs) to aid in the viral life cycle...IRE) RNA motif of the ferritin light chain RNA transcript. IRE motifs are bound by IRE binding proteins...Methods, 15, 207-212. PubMed PMID: 29400715. PubMed Central PMCID: PMC5886736. Find the RaPID Plasmids Here...

...they are not always easy to track at the transcriptional or translational level. However, several genetic... 20026026. PubMed Central PMCID: PMC2823934. Pai, Vaibhav P., et al. "Transmembrane voltage potential ...fluorescent voltage indicators (GEVI) are ideal tools to track bioelectric states (Matthew et al., 2018). These...Use of genetically encoded, light-gated ion translocators to control tumorigenesis." Oncotarget 7.15 (... (2016): 19575. PubMed PMID: 26988909. PubMed Central PMCID: PMC4991402. Levin, Michael, Giovanni Pezzulo...(2012): 313-323. PubMed PMID: 22159581. PubMed Central PMCID: PMC3243095. Additional resources on the ...

...surface display vectors to improve expression and trafficking of these biosensors.   Figure 1: Schematic...hippocampal cultured neurons, imaged 14 days post-transfection, excited at 505 nm (right). Scalebar represents...hydropersulfide-propagating enzymes 3-mercaptopyruvate sulfurtransferase (MPST) which catalyzes the desulfuration of...an enzyme-bound hydropersulfide that is then transferred to thiol-containing acceptors. These hydropersulfides...redox modification, the MPST hydrosulfide can be transferred to the roGFP2 cysteine thiols and change its ... human cell line, Hep3B. From left to right: Mitotracker Red, GFP for the biosensor, Hoechst, and overlay...signaling related fluorescence during morphine administration and withdrawal to explore how these cells may...

...students transform them into different strains of E. coli,” says Westenberg. During the transformation experiment...different genetic backgrounds of the host strains affect transformation efficiency.  Aside from the Streak Plate...add a bit of fun. He mixed together 10 E. coli strains producing different pigments, and tasked the students...students to separate out colonies from each individual strain using the streak plate technique. Time for the...single colonies, these chromoprotein-expressing strains are easily amenable to other experiments to teach...

...Interactions Difficult to detect weak and/or transient PPIs. Many PPI identification techniques rely ...often prevents the identification of weak and/or transient interacting proteins. No spatiotemporal control...BioID or Split-BioID, cells are lysed, protein extracted, and labeled protein interactors purified via ...BioID, is also useful for identifying weak and/or transient protein-protein interactions since the biotin ...speed of labeling. BioID requires 6-24 hours for tetracycline induced expression of the BirA* fusion protein...Split-BioID, Schopp et al allowed 24 hours for tetracycline induced expression and biotinylation of interacting... communications. PubMed PMID: 28585547. PubMed Central PMCID: 5467174. Find the Split-BioID Plasmids ...

...the academic science landscape: the treatment of trainee scientists as workers, at least for the purposes...purposes of labor laws, rather than as trainees in a special category, as has been the ‘labor-of-love’ philosophy... on long-term efforts to better account for and track postdocs nationwide. On this level, FoR is currently... by the development and implementation of new strategies designed to cope with current increases. These...will include a discussion on effective advocacy strategies on various levels, with panelists including: ...effectively advocating for change: from developing a strategy to distilling a message for the appropriate audience...2014): 5773-5777. Pubmed PMID: 24733905. Pubmed Central PMCID: PMC4000813. 2. Taylor, Justin Q., et al....

...the possibility of directional gene transfer, analogous to traditional restriction enzyme cloning. Sticky-end...IN THIS POST HAS BEEN RETRACTED AND FOLLOW UP STUDIES HAVE FAILED TO DEMONSTRATE GENOME EDITNG WITH THIS...and requires only one RNA rather than the two (tracrRNA and crRNA) needed by Cas9 for cleavage. Now, two...Sticky-end mediated gene transfer would be particularly helpful for targeting non-dividing cells, which are...low-frequency Cpf1 cleavage at 1-12 off-target sites; in contrast, SpCas9 may cleave at ~90 sites, according to ...the cleavage site, preventing future edits. In contrast, since Cpf1 cleaves relatively far away from the... yet characterized the potential “sticky end” transgene insertion mediated by Cpf1-generated overhangs...

...and inactivated transgene expression in neurons.  PubMed PMID: 2286602. PubMed Central PMCID: PMC3406316...sites directs the Cre recombinase to invert, translocate, or excise a DNA fragment. The availability of...creative ways. Today’s post will focus on one such strategy--the FLEx switch--which utilizes recombination...use two different pairs of target sites for this strategy to work effectively. Both loxP and lox511 are ...infected and would have the potential to express the transduced reporter gene. Generating a FLEx switch to control... orientation in the presence of Cre. A similar strategy can be employed for "Cre-off" (i.e. your gene ...O, Chambon P, and Ghyselinck NB. A directional strategy for monitoring Cre-mediated recombination at the...

...histone modifications, which can influence the transcription of genes by RNA polymerase II, or by the addition...methylcytosine in CpG islands makes  genes less transcriptionally active. The role of epigenetics in studying... before their adolescence passed on a heritable trait to their grandsons that decreased their likelihood... while still in their mother's womb passed on a trait to their granddaughters that reduced their lifespan...of tumor suppressor genes can repress their transcription and inactivate them. Global hypomethylation,...restriction enzyme digestion, DNA adenine methyltransferase identification, and bisulfite sequencing. ...Essentially this reporter construct consists of one substrate (the histone-derived peptide) which can localise...

... scientific training was put to use as we explored ideas, planned out potential strategies for pursuing...with a similar conundrum – she had the skills, training, and motivation, but there very few jobs available...our ideas got pretty good. We started doing extracurricular research on some of them to see how feasible...SynBio Axlr8r We limped along with very little traction for quite a while, and even had some serious self-confidence...and another $30,000 in mentorship and business training. The money is a drop in the bucket against the... the technology we will need, and the business training to make the most of it. It has also allowed us...below. We’re happy to answer questions about this transition (so far) and what we’ve learned.  Thank you ...

...expensive exogenously-added substrate, chromoproteins don’t require other substrates for screening. Thus chromoproteins...visible light to give colors in ambient light. In contrast, the widely used green fluorescent protein (GFP...bright green fluorescence when exposed to blue to ultraviolet light, whereas chromoproteins do not fluoresce...anemone’s red colored tentacles resemble worms which attract fish for them to prey upon (Figure 1). Figure... a gene encoding a blue chromoprotein, after transforming them into your cells, you can simply look for...imaging microscopy-based Förster resonance energy transfer (FLIM-FRET) in living cells (Figure 4). They created...by modifying the surface of the chromoprotein Ultramarine with hydrophilic amino acids to reduce non-specific...

...to find the ones you’d like to revisit. Strategically traverse the sea of posters If you come prepared...interest causes cancer, as well as therapeutic strategies to target it, than you typically would at a microbiology...keep you energized throughout the session, bring extra clips to hold up your poster, and pack a few business...seconds before the session begins. Plus, you’ll have extra time to practice at the actual venue. Introduce ...For most larger conferences, poster titles and abstracts are available online or in an app prior to the... through the list at the session so you can strategically use your time. Don’t have access to a list of...in to thirty-minute-long presentations, which constrains the number of posters you can see. If you are...

...wavelengths (See this handy emission spectra chart to find your antibody's spectra). Your cytometer will therefore...probes on the antibodies may have similar emission spectra, meaning they will emit fluorescent light at similar...be compensated for one another. This overlap in spectra is often called "spillover." For example, if your...BV510 and AmCyan have almost identical emission spectra but may be called BV510 on one cytometer and AmCyan...against APC-Cy7, the populations are in more of a straight line and are only positive for APC (Figure 1B)... plot in 2A, the populations are not perfectly straight, but the center of each population is in a line... line in the plot. However, because we can see straight lines in the populations in the other corners ...

...IN THIS POST HAS BEEN RETRACTED AND FOLLOW UP STUDIES HAVE FAILED TO DEMONSTRATE GENOME EDITING BY THIS...used by respondents (transfection method, amount of each component per transfection etc.). Take Pooran'...an ssDNA guide. Therefore, researchers must co-transfect cells with 5’-P-ssDNA guides and an NgAgo expression...delivery methods. Researchers can conveniently transfect/inject cells with in vitro preassembled Cas9/gRNA...researchers will have to carry out repeated transfections with the ssDNA guide, although many may find... an ssDNA guide. It is possible that, during translation, only a small fraction of nascent NgAgo protein...2014): 1262-1278. PubMed PMID: 24906146. PubMed Central PMCID: PMC4343198. Resources on the Addgene Blog...

...Melanie Fox, founder and executive director of Central Indiana Science Outreach and a Postdoc at Indiana...student culminated in my founding a nonprofit called Central Indiana Science Outreach, or CINSO. We organize...experiences in grad school, though often called “nontraditional,” helped prepare me to start CINSO. Here, I... from these contacts, I would have quit my “nontraditional” career path long ago. Use your analytical ...an excellent resource that has gained a lot of traction with researchers. It is an excellent way to meet... board. Although my goals may not have been “traditional,” I was able to fully take advantage of my graduate...currently the founder and executive director of Central Indiana Science Outreach and a Postdoc at Indiana...

... muscle contractions, and the control of voltage gated ion channels all depend on transmitting and sensing...insufficient level. The heartbeat – a muscle contraction – is directly related to the electrical impulse...propagated through multiple cells to do things like translate perceived signals to physical actions. Understanding...rhodopsin proteins were initially evolved for ion transport, not fluorescence, they are not nearly as bright...conditions, which are necessary conditions for penetrating deeper tissues depths (1 mm), important for certain...and emit signal from. This specific example is attractive due to the large number of sheer molecules and... this method have been to tag, cage, uncage, and trap the chemical fluor with the genetically encoded ...

...but they can also be expressed in bacteria and extracted with common protein purification methods. Read...below for a comparison of nanobody, HCab, and traditional IgG antibody structures. Functional comparison...microscopy. Their size also limits their ability to penetrate tissue. Because antibodies are large and bivalent...recombinant; Require animal sacrifice Poor tissue penetration Less resolution due to larger label displacement... do not require animal sacrifice Good tissue penetration Can be labeled with multiple dyes Greater resolution...Their small size also allows for better tissue penetration and decreases the distance between a fluorescent...Lee, C., Pleiner, T., Schliep, J.E., Stark, H., Trakhanov, S., & Urlaub, H. (2015). Nanobodies: site-specific...

...C-to-U and A-to-I transcriptome-wide off target effects observed in whole-transcriptome RNA-sequencing, ...RNA editing has multiple advantages over more traditional DNA editing systems; first, RNA editing doesn't...would convert adenine to inosine, which the translational machinery treats like guanine. This RNA editor...amount of off-target editing. In one direct transcriptome-wide comparison, REPAIRv2 reduced off-target... on the ADAR2DD residues that contact the RNA substrate resulted in 15% C-to-U editing activity. They ...the RESCUE system’s ability to edit endogenous transcripts finding up to 42% editing efficiency. They note... (2016):aaf5573. PubMed PMID: 27256883. PubMed Central PMCID: PMC5127784. Find plasmids from this paper...

...control strain. A good negative control strain is an untransformed culture of the same strain of bacteria...screening colonies for the presence of an insert. Traditionally this has been done with restriction enzyme digest... colony PCR is designing primers. There are 3 strategies for primer design: 1) insert-specific primers...mutations.  There are lots of different cloning strategies, but regardless of which is your favorite, colony...control. A good positive control is bacteria transformed with the same backbone plasmid. If this control...Cloning Technique Learn to Perform a Bacterial Transformation Deposit Your Positive Clones with Us ...