...fluorescent protein (XFP) genes in a transgene. When multiple copies of the transgene cassette are introduced, stochasticity...delivery is more common than conventional transgenic strategies. In such vector-based gene delivery systems... XFPs in the original method, we used the Tetracycline trans-activator system to boost the expression ...expression levels, and we named the system Tetbow (Tetracycline trans-activator Brainbow) (Sakaguchi et al., 2018...Bio-Protocol 8 (2018): e3046. Livet, Jean, et al. "Transgenic strategies for combinatorial expression of fluorescent...troubleshooting your Tetbow experiment Plasmid concentrations and AAV titer - The XFP genes have to be introduced...highest color variations. Adjust the plasmid concentrations or AAV titer for the best results. The timing...

...with a 3-part strategy: Train: Nearly every new Addgene employee gets “Lexicon Training.” Since the lexicon...you can be incredibly frustrating. Clarifying the words —> decreasing frustration —> increasing the happiness...and racks meant we had fewer errors in our LIMS tracking system. Though you might not have to communicate... lexicon is a “living” document, I update the training frequently. In hindsight, getting a list of frequently...resources on the Addgene blog Read about other transferable skills Find all of our science career related...

... the non-edited strand to encourage the cell to repair the DNA using the edited strand as a template —... ever lose track of which acronym is which. Acronym Full name Notes   Base transition Base change...our last post, we talked about the first base transversion editors: CGBEs, or C → G Base Editors. CGBEs...editor could excise other bases? Could other transversion editors be close at hand? Once again, this post... the end of this post! Figure 1: Base transitions are edits between the purine bases (adenine, ...bases (cytosine, thymine, and uracil). Base transversions are edits that convert a purine to a pyrimidine... Inosine excision leads to the first adenine transversion editors Uracil excision is the most common type...

...emission spectra for BV421 and BV711 on a 3 laser spectral flow cytometer   Using spectral flow When...autofluorescence extraction feature. Although autofluorescence extraction is an optional feature of spectral flow ...portion of that spectra to use for compensation.     Figure 1: The emission spectra and filters ...cytometer.   Spectral flow cytometry Full spectrum flow cytometry, or “spectral flow” cytometry, ... be distinguished using spectral flow since the machine uses the full spectral signature, not just a narrow...in the spectra can be used to distinguish these two fluorophores from one another during spectral unmixing...with spectral flow—you can run a three color panel or a 40 color panel! When preparing a spectral flow...

...Enhancer traps Like promoter traps, enhancer traps lack enhancer elements and require the trapping of endogenous...circumstances, trap methods may be the preferred screening option. Promoter traps Promoter trap systems lack... enhancer traps! However, don’t count the other traps out just yet. Gene and promoter traps unambiguously... up on their modern counterparts. What are traps? Traps – gene, enhancer, and promoter – all rely on...in fly, mouse, and yeast models. Why traps are useful Traditional genetic screens employ various on/off...expression of the gene they monitor. Gene traps Gene traps are promotor-less constructs that contain ...gene product resulting in a transcriptional fusion, much like promoter traps. While this system does not...

...DEAE-dextran can improve transduction efficiency. In addition, this study found that transduction efficiency...passage number of a culture the lower the transfection and transduction efficiencies. Cultures should only be...others opt for a single harvest after transfecting with the transfer and packaging plasmids. A benefit of...type may not translate well to other cell types. To overcome this issue, try transducing with a range ...Zhang, X.Y., Reiser, J.. "Production, concentration and titration of pseudotyped HIV-1-based lentiviral...vector titration and in the use of multiplicity of infection (MOI) for predicting gene transfer events... for getting the most out of your lentiviral transduction experiments. Love your cells and they'll love...

...E. coli expression strains  Note: All strains are derived from the E. coli B strain, except ** which are...induction strain for example, nor will a p15 plasmid be compatible with a pLys strain. Your strain may require...popular commercial strains designed to avoid them. Why do I need an expression strain? Protein expression...below, certain specialized strains are available to confer greater transcriptional control, assist with proper...most expression strains to accommodate high protein levels including:  ompT: Strains harboring this mutation... These strains have an inactivated native restriction/methylation system. This means the strain can neither... K12 Strain Resistance Key Features Genotype Use BL21 (DE3)   Basic IPTG-inducible strain containing...

...emotions, such as frustration or anger, because your discussion will get side-tracked, and you might create... your project and work hours. While specific strategies to deal with each of these personality types ...these principles it would be “assertiveness.” 7 strategies to become more assertive & resolve conflicts ...supervisors are difficult people. The following seven strategies will help you to become more assertive and resolve...between you and your supervisor, you need to demonstrate that they can count on you to follow through ...professional conflicts. At the workplace, these strategies will help you to communicate with confidence ...your supervisor is a difficult person, the above strategies will still be helpful but you will need to be...

...the E. coli B strain. Common E. coli strains used in the lab Most of the commercial strains you find today... the intestinal tract of animals. There are many different naturally occurring strains of E. coli, some...commercial lab strains of E. coli used today are descended from two individual isolates, the K-12 strain and the...the B strain. K-12 was isolated from a patient in 1920 and eventually led to the common lab strains MG1655...1918 but was first referred to as “B strain” in 1942. The BL21 strain and derivatives are the most common...changes found in E. coli strains. Table 1: Common gene mutations found in E.coli strains Gene(s) Description...of the popular strains, their genotypes, and their primary use in the lab. These strains are all based ...

...terminators and resistance cassettes for selection of transgenic plants. The complete GreenGate toolkit, now available...the time required to generate multi-construct transgenic plants. In the most practical terms, what GreenGate...comparison to more tedious and time-consuming traditional cloning methods, Lohmann said. The key component...like other endonucleases except that they cut at arbitrary sequences a defined distance away from the recognition...principle work in any plant species. Tricks of the trade Lohmann does have one special tip for anyone looking...efficiency, simply stopped working. They finally traced the problem back to the endonuclease. “It’s just...about it but be gentle. If it no longer works, just trash it.” Lohmann and his colleagues including Joachim...

...lab. Extracurricular activities don't have to be ‘extra’ Today’s graduate and postdoc training focuses...using multidisciplinary, collaborative, and translational approaches for scientific tool and technique...However, careers outside of the lab highlight “transferable” skills such as leadership, management, and ...in a myriad of professions is to engage in extracurricular activities (EA). EAs can: Introduce new perspectives...may expose which experiments are crucial in demonstrating the true innovation, impact and, in turn, the...opportunity to expand associations outside of the traditional academic setting and to shape and strengthen ...strengthen interests and skills. Although the word “extracurricular” may be taboo in the lab, multiple research...

... don’t wait. Start communicating now. These 9 strategies will get you started:  Subscribe to Career Blog...internships to cover their annual meetings. 8) Training – Get a formal education Some people get into ...journalism graduate schools where you may take a science track. If you don’t want to commit time and money to a...engagement for institutions and the Royal Society gives training in media skills, writing about your research and...learning courses at the National Council for the Training of Journalists (NCTJ) or the Alan Alda Center ...

...way to begin the search is by using selection strategies, where only cells that have gained or lost a ...plasmid cloning.  But oftentimes, a selection strategy is not enough on its own to help you find your...plasmid. In many cases, you will need a screening strategy as well. In a screen, you aren’t killing off a..., then screen Why add a screen to your cloning strategy? A screen will help you more easily identify successful...information (more on this below). Plating the transformation on ampicillin plates containing X-gal allows...Fortunately, for the lab researcher, when the substrate X-gal is broken down by 𝛽-galactosidase, it turns...blue-white screening at Addgene! Some bacterial strains contain lacZ in their genome, and scientists have...

...repeats (ITRs) are what makes an AAV transfer plasmid an AAV transfer plasmid. ITR sequences are 145 bases...read through this challenging sequence. Transgene The transgene is the gene that will be delivered by the...Most AAV transfer plasmids have a promoter, but this element isn’t required when the transfer plasmid ...control transgene expression. Lox sites are directional 34 bp sequences that can flank the transgene and ...(WHP) Posttranscriptional Regulatory Element (WPRE) forms a tertiary structure when transcribed which ... nuclear export of RNA and RNA translation, and promotes RNA transcript longevity. The polyA is located...proteins needed for the virus to replicate,  and the transfer plasmid which contains the viral genome. In today...

... as a manager.   Strategies for a successful transition To successfully transition into a managerial ...Understanding the transition While a PhD equips you with exceptional technical skills, transitioning into management...exploring alternative career trajectories within the industry, but the transition wasn’t easy - I had to reinvent...Stories and Strategies for PhDs Making the Leap to Industry." He successfully transitioned to industry... most pivotal decisions many PhDs face when transitioning from academia to industry is whether to stay...involves a different skill set: mentoring, setting strategic goals, and fostering team success. This shift ... managerial role. Along the way, I discovered strategies to effectively pivot my career development and...

...contain plasmids deposited at Addgene from the abstract page. Check out that beautiful Addgene “Find Plasmids... Plasmids” badge in the image below. If an abstract on PubMed has the “Find Plasmids” badge, you’ll know... your PubMed account and are browsing article abstracts you should see the “Find Plasmids” box on papers...

...imaginations to use in this world where we are often trained to follow protocols, guidelines, and standards.... an odd or quirky lab story you’d like to see portrayed in LEGO form, feel free to share with us at BrickingScience...

...CRISPR-associated Transposase) by the Zhang group. Mechanism of CRISPR-transposons One of the CRISPR-transposon systems...DNA transposons have been hindered by their limited programmability: depending on the transposases involved...-guided transposition. By natively combining the DNA integration capabilities of transposases and the ...crRNA.  Three transposase proteins (TnsA, TnsB, TnsC). The donor DNA (aka mini-transposon), which contains... each type of CRISPR-Transposon system. (B) Mechanism of RNA-guided transposition in type I-F INTEGRATE.... Separately, TnsABC forms a transposase complex with the mini-transposon, excises it from the donor molecule...characteristic of the Tn7-family of transposons. Insertion of the mini-transposon can occur in two different orientations...

...and Harvard Medical School. Scientists making transgenic organisms with Cas9 should be aware of the potential...conducted with extreme caution. Why does making transgenic organisms with Cas9 risk creating a gene drive... there are many easy-to-implement confinement strategies that are robust to human error. Used in combination...these robust and easy-to-implement confinement strategies2. Our hope was that anyone else who came up with...improve safety. What safeguards and confinement strategies are available? Molecular confinement involves...drives that can spread through populations of transgenic laboratory organisms but not wild organisms. ...targeting a synthetic sequence will only spread in transgenic laboratory populations with that sequence2. Both...

...body, like the gastrointestinal tract. Require Administration of Substrate: Some luciferase reporters, such...therefore produce signals in ultrasound imaging. Ultrasound is an attractive imaging platform for in vivo...coli aided in ultrasound imaging of the mouse gastrointestinal tract. The E. coli strain Nissle 197 (ECN...NanoLuc, require injection of substrate to produce bioluminescence. Substrate pharmacokinetics and biodistribution...because  ultrasound devices are inexpensive, widely available, and have deep tissue penetration and high...are invisible to ultrasound, which allows for differentiation of arg1 and arg2 ultrasound signals. This ...because it’s a probiotic strain that can colonize the mammalian intestinal tract and has been used for 100...