...finally decide the LEGO laboratory was good enough. There are no pre-made LEGO laboratory sets so we had to ...to show people the very human struggles in the laboratory routine. We hope that sharing and laughing at...the LEGOs were mirrored by the nearly infinite regulatory capabilities of gene pathways just waiting to...was creating a LEGO stage that resembles a real laboratory. It only took one night to build the first model...use our imaginations to mold it all into a full laboratory. It is priceless to see our “toys” gaining life...working as scientists in a custom made, colorful laboratory. When we started, the hardest part of the process...building each scenario, I go “WOW, now I have my own laboratory” (the main character is much more than a piece...

...Despite its relatively recent development, the history of optogenetics begins almost 50 years ago with...Wick and Krook-Magnuson, 2018). Optogenetic FLEx vectors contain a strong promoter upstream of an opsin ...Cre-dependent viruses, such as the optogenetic FLEx vectors in combination with transgenic animals or rAAVs...example, Taylor and colleagues used rAAV FLEx-based vectors to study the neural circuitry behind the mechanisms...paper, de Jong and colleagues used rAAV FLEx-based vectors to answer this question, mapping and characterizing...spatio-temporal precision and reversible modulation of FLEx vectors combined with the use of several Cre transgenic...motivated behaviors (de Jong et al., 2019). FLEx vectors represent, therefore, an ideal partner to optogenetics...

... learning. 1. Create a peer mentoring group We don’t all get the mentoring support we need, and most of...to access great mentorship is by recruiting your own peers and classmates! Your mentors don’t need to be...Own Peer Mentoring Group: A How-To Guide for Scientists.   Once in-person, peer-mentoring groups ... of us could use more mentors to listen, guide and support us during training and throughout our careers...goals.  I’ve been surprised to see that virtual mentoring groups seem to be as successful as in person groups...? You could do a talk virtually for your peer mentoring group (see above). However, now that we are all...initiative. In fact, hear from Addgene’s former blog editor Tyler Ford on how initiative landed him at Addgene...

...transcriptional regulator to modulate expression at that locus. The use of regulators allows a greater...shifted in recent years toward tinkering with regulatory networks and multiplex gene control. However,...approaches are often met with the challenges of regulatory bottlenecks and their scope is limited by the...Far removed from the bio-industry’s vision of ‘biofactories’, most successes in metabolic engineering have...us one step closer to the vision of metabolic biofactories by demonstrating the use of CRISPR-based RNA...scaffolds that can recruit various transcriptional regulators (Zalatan et al. 2015).  These scaffolds are stretches...levels dependent upon the type of transcriptional regulator or the number of designed scaffold binding sites...

... at some of the stories behind the names. We also have many plasmids in our repository that you can use...to a specific set of DNA-binding transcription factors to repress the expression of genes primarily involved...Cancer Institute! Sonic Hedgehog, an important regulator of embryonic development The largest conference...the hedgehog signalling pathway, which is a key regulator of all bilateral animal development. SHH plays...publication, Ivics et al 1997. Spaghetti Squash, a regulator of cellular contraction You may think that a gene...vessels). Spaghetti squash (Sqh) is the Drosophila Regulatory Light Chain (RMLC) of nonmuscle myosin II (Karess... Jordan and Karess, 1997), which contains the regulatory regions that are necessary for myosin activity...

...exactly how postdoctoral salaries are changing. Transparent salary information for postdoctoral positions...institutions set their postdoctoral salary scales to the NRSA scale (Figure 21, National Postdoctoral Association...was contributed by Future of Research Executive Director, Gary McDowell. On Dec 1st, the threshold at which...when the ruling was issued, a joint blog post by Director of the NIH Francis Collins and Secretary of Labor...paid. Institutional salary policies are one indicatator of how salaries may changes due to the FLSA. ...as having postdocs, representing ~85% of the postdoctoral workforce. Based on our communications with ...according to the NSF data, account for 18% of the postdoctoral workforce in the sciences. The Dana Farber Cancer...

...the activator - the CRY2 photolyase homology region (CRY2PHR) fused to a transcriptional activator domain...directed by the guide RNA (gRNA), while the CRY2PHR-activator fusion floats freely, depicted in the figure below...and CIB1 proteins heterodimerize and move the activator into position to activate gene transcription. ...manner. With their light-activated CRISPR/Cas9 effector (LACE) system, the Gersbach lab utilized a similar...fusion of full-length CRY2 and the transcriptional activator domain VP64. Using this system in HEK293T cells...photoswitchable proteins derived from the fungal photoreceptor, Vivid (VVD, N. crassa) (Kawano et al., 2015...transcription, they fused BldD to the transcriptional activators p65 and VP64 which in turn transcribes N-Cas9...

... and progenitor cells. Now with the DAP array, it is possible to use the state-of-art base editor, prime...prime editor, or any CRISPR-based gene editors, for correcting mutations or installing functional genes...thought to enable MBE by using Cas12a fused base editors (dCas12a-BE). However, the poor editing efficiencies...they found led them back towards Cas9 fused base editors (nCas9-BE), as Cas9 is mechanistically more favored...number of gRNAs that can be packaged into viral vectors for potential in vivo applications or multiplex...array.7 Using DAP arrays with a variety of base editors, the Gao Lab successfully demonstrated up to 31...unprecedented number of genes with a single viral vector.   Fig. 1: Schematic of multiplex gene ...

...adjacent fragment or vector. 5′ overhangs are generated on all the fragments and the vector, specifying the... together through transformation, so the cloned vector must be ligated. But in LIC, longer single-stranded...due to the absence of the other dNTPs. Once the vector and insert are (separately) digested to create ...received an important update, courtesy of Addgene depositor Stephen Elledge. His new method, named sequence...homologous recombination. SLIC inserts and linearized vectors must have homologous DNA sequences (20–60 bp) on...complete plasmid. Besides being compatible with any vector, the SLIC protocol itself is flexible. For one ...digestion (no dNTPs needed), allows both insert and vector to be digested in the same tube. Alternatively,...

...calcium indicator (Shemetov et al., 2021). In addition to FRET, intensiometric calcium indicators NIR-GECOs...proteins (NIR FPs) were derived from natural photoreceptors incorporating biliverdin IXα (BV) autocatalytically... derived from bacterial phytochrome (BphPs) photoreceptors have higher apparent cellular brightness than...were derived from cyanobacteriochrome (CBCR) photoreceptors (Oliinyk et al., 2019). They were engineered...performance, and applications of near-infrared calcium indicators in this recent review (Shcherbakova et al., 2021...encoded near-infrared fluorescent calcium ion indicator. Nat Methods 16, 171-174 (2019). Matlashov, M..... A near-infrared genetically encoded calcium indicator for in vivo imaging. Nat Biotechnol 39, 368-377...

...genes. We’ve created enough regulatory elements, CRISPRs, repressors, activators. We’ve been putting together...CRISPR/Cas genetic circuits are linked to the regulatory networks within E. coli host cells. ...in building genetic circuits is getting enough regulatory proteins that don’t interfere with each other...programmable that you can make a very large number of regulators. That basically means larger genetic circuits...that could conceivably be as large as natural regulatory networks. Addgene: What kinds of things could... pattern, shape. All of that is controlled by regulatory networks in the plant. If we want to access that...CRISPR is how much easier it is to work with than regulatory proteins. There are still things we haven’t solved...

...Looking for a mentor or want to start a peer mentoring group? "What Makes a Good Mentor?" Form Your Own...form a peer mentoring group to get real feedback Write whenever you can, find supportive editors so your ... Own Peer Mentoring Group: A How-To Guide for Scientists Download our eBook: Mentoring for Scientists ...your PhD or perhaps you have almost completed a postdoctoral position… or two. You have learned a lot. Whether... scientists, create and stick to timelines Be a mentor in a formal program Start something (a journal,... plan a conference Recruit many supporters and mentors that will speak for you (and you for them) Communication...

...Mice can be engineered by inserting a targeting vector containing the gene of interest into a zygote, ...microinjection of DNA or infection with a viral vector containing the gene construct can be used to insert...generated via homologous recombination. A targeting vector is electroporated into embryonic stem cells and...engineer knockin mice. Co-injection of a targeting vector containing homologous ends to the breakpoint promotes...promotes homologous recombination of the targeting vector. CRISPR/Cas9-based systems have been developed ...the expression of a particular gene. Targeting vectors used to create knockout mice include a reporter...standing technique is the injection of a targeting vector into embryonic stem cells, which are then injected...

...massive amount of information regarding which regulatory proteins, signaling molecules, mutations, and...little is known about the order or timing of these factors. Recombinase-based state machines (RSMs), which...In the absence of extra recombinase-specific co-factors (which should be excluded from the system), these...paper (1). Lastly, it is possible to build gene regulatory RSMs (GRSMs, the subset of RSMs that regulate...also demonstrates that genetic parts (genes, terminators, and promoters) may be interweaved onto a GRSM...parts used in this paper (recognition sites, terminators, promoters, and genes) were built with flanking...them. We recommend using strong, directional terminators and promoters with buffer sequences on their ...

...following: the elevator pitch, talks, the poster session, networking, and social media. The elevator pitch Before...research, gathered up your data into an exciting story, and are ready to present your findings at a conference... Before the conference Practice your “elevator pitch,” the 30 - 60 second blurb you will use to introduce...work you are familiar with. You can tweak your elevator pitch slightly if you know a bit more about the...or talk. After the conference After using your elevator pitch several times at the conference, you might...might notice some trends. Were there parts of your elevator pitch where the other person seemed confused, ...questions. If you or your lab identifies a potential collaborator from the meeting, reach out to them, pointing...

...: A knockout targeting vector designed to insert a resistance gene. The vector contains a neomycin resistance... homologous sequence using a specially designed vector that contains sequence homology to the locus of... of the targeted gene have been cloned into the vector on either side of an antibiotic resistance gene...Figure 2). Figure 2: A knockout targeting vector designed to insert and later remove a resistance... the sequence to be inserted is cloned into the vector between the homologous sequences together with ... Figure 3: A knockout/knock-in targeting vector designed to insert GFP into a given locus. ...earlier. In this case, you design your targeting vector such that a set of three LoxP sites flank the resistance...

...the tool, and develop new applications for magnetoresponsive proteins. Motivated by curiosity Although...Maria dreamed about the possibilities of a magnetoresponsive fluorescent protein. Controlling an optogenetic...experiments to see if any small molecules act as a cofactor (among others, flavins are very effective) and...self-labeling FlavinTag made a reliable one-component magnetoresponsive system that could be expressed and purified...thrilled to see that MagLOV’s “astonishing” magnetoresponse was clearly visible whether expressed in E....or in purified form, and didn’t need any added cofactors.      Figure 2: Fluorescence intensity...respond to magnetic fields?” and “What are magnetoresponsive fluorescent proteins good for?”, and while...

...Bacillus subtilis and the yeast Pichia pastoris. The improved secretory capacities of these microbes make ...this information, you can then pick an expression vector and expression system. If you find that your protein...not fold well, you should screen a few different vectors to see which gives you the most functional protein...coli: purifying from Bacillus subtilis and Pichia pastoris pCri also includes plasmids designed for protein...producing difficult-to-fold proteins. In addition, P. pastoris has the capacity to glycosylate proteins and may...peptide (SP) that routes the protein through the secretory pathway to improve folding and disulfide bond ...coli, the use of a thioredoxin A (TRX)-containing vector can improve disulfide bond formation. pCri also...

...include the laboratory personnel, the serum, the cell culture media, water baths, incubators, etc. Contamination...another. Multiple cell lines should not be stored in a single incubator since it increases the risk of cross-contamination... Addgene's viral vectors articles Find tips and tricks for working with viral vectors Resources on Addgene.org...Contamination! Tissue culture media often have indicators included in the media that turns yellow from ...proliferating shortly. Once they are present in the laboratory environment, they are difficult to eradicate....using expired products or reagents that have been stored for a longer time. Use fresh media as much as possible...tighten the flask cap before placing them in the incubators. This will also solve the problem of contamination...

...petunia SL enzymatic receptor DAD2. SLs induce a conformational change of the receptor which modulates the...subset of the new plasmids and viral preps in the repository through our hot plasmids articles. These articles...inducible CRISPR inhibition (CRISPRi). A) Lentiviral vector design. B) Strategy for leak-free transcriptional...detection of SLs. The authors also showed that the receptor-based fluorescent biosensor has a high sensitivity...biosensor based on the parasitic Striga hermonthica SL receptor HTL7. This novel type of biosensors could be used...  Design and mechanism of action of the receptor-based SL fluorescent biosensor. Image from Chesterfield...CRISPR plasmids are always being added to the repository. To find all of the CRISPR plasmids available...