...host range plasmids. FEMS Microbiology Letters, 348(2), 87–96. https://doi.org/10.1111/1574-6968.12241  ...mobilization of broad host-range IncQ plasmids. Plasmid, 62(2), 57–70. https://doi.org/10.1016/j.plasmid.2009.05.001...negative hosts. Biotechnology and Bioengineering, 106(2), 326–332. https://doi.org/10.1002/bit.22695  Schmidhauser...Understanding the characteristics of natural plasmids and how they function in nature can inform us on...trichosporium, Myxococcus xanthus, Neisseria spp., Paracoccous denitrificans, Pseudomonas spp., Rhizobium spp...Moraxella spp., Mycobacterium aurum, M. smegmatis, Paracoccus denitrificans, Pasteurella multocida, Porphyromonas...Escherichia coli, Gluconacetobacter xylinus, Paracoccous denitrificans, Pseudomonas fluorescens, P. putida...

...Brainbow mouse neural labeling system. Brainbow-1 and -2 have been further refined to create Brainbow 3.2. ...M-CFP expression; these deletions are defined using 2 loxP variants (only identical loxP sites can mediate... each fluorophore. Third, Brainbow-1 and Brainbow-2 each contained a “default” state; for example, Brainbow...processes not previously visible with Brainbow-1 and -2. The general structure of Brainbow-1.0 is retained...fluorescent proteins to find those with ideal characteristics (low aggregation, high photostability, and ...

...is unique compared to the rest of the genome and (2) the sequence is located just upstream of a Protospacer...pyogenes Cas9 variants with varying PAM sequences, 2) the use of Cas9 homologs derived from species other...Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. Cell 163:759–771 . https://doi.... PAMs, respectively (Walton et al., 2020). Characterization of Cas9 from additional bacterial species ...additional Cas9 species PAM sequence may not be characterized Non-SpCas9's bind a variety of PAM sequences.... Furthermore, non-SpCas9's may have other characteristics that make them more useful than SpCas9. For...

...our deposit spreadsheet to complete Step 1. Steps 2 and 3 are usually very easy — our tech transfer team...columns are shown in red in the spreadsheet (Figure 2) and are described in Table 1. Some cells have drop-down...Filling out and sending the deposit spreadsheet Figure 2: Example of the deposit spreadsheet showing the first... Answer “What does this plasmid do?” in <200 characters. A descriptive purpose focuses on the practical...

... intensity of the cell’s illumination (see Figure 2). H describes the maximum signal strength, while W...SSC-A, so most people use that setting. Figure 2: As the cell passes through the laser beam and causes...scale in proportion (while H and W do not; see Figure 2), single cell events will show up in a straight line...marker expression, and viability. Gates are the parameters the machine uses to differentiate between variations...bit of theoretical background on the different parameters that your gating strategy will be based upon.... and Area Within FSC and SSC, there are three parameters that are being measured: height (H), area (A)...events. You are free to choose any size-related parameter, such as FSC-H, FSC-A, SSC-H, SSC-A, etc. You’...

...target sites for editing. PAM sequences can be from 2-6 base pairs in length and follow the DNA sequence... Eric Perkins Mammalian genomic DNA is not just a 2-D linear molecule snaking through our cells’ nuclei...publicly available metagenomic data including the Tara Oceans Project datasets. This study demonstrates...fused dCas9 to a truncated CIB1 protein from Arabidopsis (CIBN), which they call the LADL Anchor. Using... iterations is the addition of CRY2, another Arabidoposis protein that heterodimerizes with CIBN upon ...

...portion of our careers. In that time we may have only 2-3 formal supervisors that will provide guidance and...be challenging – is advice and guidance from only 2 people enough?  For many years I’ve been organizing...lighthearted, the article does highlight key characteristics of a good mentor: Yoda image courtesy...

...type. Find some transduction characteristics of select AAV serotypes in Table 2 from the paper. Delivery ...routes for neuroscience see Table 1 from the paper. 2. Manipulate your cell of interest: Cell-type specific...

...additional rounds of panning and scFv characterization.    Figure 2: Phage display starts with a pooled...the short epitope GCN4, also called a scaffold; and 2) a GCN4 binding scFv that’s fused to GFP. When the...Fv (Variable Domain) (scFv) Fragments. Antibodies 2:193–208 . https://doi.org/10.3390/antib2020193  Khantasup...another decade when in 1988 they were cloned by two separate labs (Bird et al., 1988; Huston et al., 1988)....., 2012, Bates and Power., 2019): rapid blood clearance, which is useful for imaging applications; better...small size (Bates and Power., 2019). Their rapid clearance from blood can be a drawback for therapeutic applications...Khantasup K, Chantima W, Sangma C, Poomputsa K, Dharakul T (2015) Design and Generation of Humanized Single-chain...

...digestion of the destination vector with SapI yields 2 sites- the first site accepts the sgRNA target sequence...marker, and connector to be released (Fig. 1, Step 2-3). Since donor plasmids of the same type will produce... a gRNA vector that targets the gene of interest, 2) a plasmid to specify the reading frame of the insertion...can be modified to assay additional protein characteristics. For example, Masato Kanemaki's lab has developed...

.... Let's look at an experimental example in Figure 2. Figure 2A shows the sample prior to compensation.... line with the other populations.   Figure 2: Compensation example based on experimental sample..., you will see the populations move, as in Figure 2, following compensation. Figure 3: Flow plot...can correct for this spillover and more easily separate out your target population. How do I know if ...

...resolved loci with physical map distances of 75 and 2 Mbp, with the calculated fluorescent distances correlating...established physical map. In comparing pairs of targets ~2 Mbp apart, they noticed that they could evaluate the...intrachromosomal loci, a major benchmark in characterizing the 4D nucleome. Future modifications and applications...

...the system outlined in Schmid-Burgk, J.L., et al (2). In this post, I’ll cover two great applications ...molecularly evolved from mNeptune2 through 33 mutations and 2 deletions. CyOFP1 has a higher quantum yield, brightness...luciferin 24 hours later demonstrated a more than 2-fold brighter signal from Antares as compared to Fluc...PMID: 22894855. PubMed Central PMCID: PMC3501149. 2. Schmid, J.L., et al. (2016) CRISPaint allows modular...create a calcium sensor with the following characteristics: No photoexcitation High Ion specificity High...

... is the genetic test used for sickle cell anemia (2). Southern blotting, in conjunction with restriction...program) can then reproduce the DNA sequence (Figure 2). Accuracy and accessibility led to the widespread...biology98.3 (1975): 503-517. PubMed PMID: 1195397. 2. Chang, Judy C., and Yuet Wai Kan. "A sensitive new...these reactions out in separate lanes of a gel. Since the sequencing gel will separate the DNA based on size...instead use methods like pyrosequencing that are parallelized to sequence thousands of reads at the same time...Detection of specific sequences among DNA fragments separated by gel electrophoresis." Journal of molecular ...

...into the several steps outlined below and in Figure 2. For a deeper dive into the steps and techniques, ...mounting sample on slides, and confocal   Figure 2: High-level overview of an indirect immunofluorescence...recognize the species of your primary antibody and 2) is conjugated to a fluorophore that suits your experiment...depend on the target and sample type. Sample Preparation This step can involve multiple smaller steps...Preservation and Imaging Similar to Sample Preparation, this step can involve multiple smaller steps...

...phages but to also destroy the phage upon re-exposure (2, 3). More specifically, specialized Cas proteins snip..., accessed 25 November 2013. Author: James Atmos (2). Many CRISPR/Cas moieties carry out these functions...length and paste them into the CRISPR sequence. Separate Cas proteins then express and process the CRISPR...of more than 40 cas gene families have been characterized. To date, 83% of archaeal and 45% of bacterial...

...one injection step! The entire protocol takes about 2-3 weeks but requires less than eight hours worth of...for C. elegans genome modification. Figure 2: Schematic from Dickinson et al. (2015) illustrating... is inserted at the 5’ end of a gene, the SEC separates the targeted gene from its promoter, creating ...Dickinson et al. tested this system across 7 genes in parallel; when they injected 60-80 animals per gene, they...

...parts: (1) β-strands 1-9 (β1-9) and the one ɑ-helix, (2) β-strand 10 (β10), and (3) β-strand 11 (β11). Each...activating caspase-3, and detected fluorescence within 2 to 5 hours, a time consistent with previous methods...necessary for fluorescence. When these parts are separated from each other, the protein cannot fluoresce....fluoresce. When β10 and β11 are linked together, but separated from β1-9 they will rapidly recombine with β1-9...on protease activation. Normally, β10-11 are antiparallel. To prevent binding, the β strands in FlipGFP...FlipGFP were redesigned so that they are parallel to each other and thus can no longer fit with β1-9. They...dimerize together and force the strands into a parallel conformation. Between β11 and one of the linkers...

...is always best to have options.      Fig. 2: Addgene’s western blot for mEGFP-HRas (from Plasmid...the presence of oxidizing agents. Adapted from Fig. 2 Campbell et al, Nature Methods   Find YFP plasmids...Susanna Stroik The Doudna lab’s recent work characterized a new tool for RNA targeting, with a leg up ... multiple antibodies allows you to use them in parallel to help amplify your signal - giving you the benefit...observed high fluorescence resilience to these preparations. After crystalizing the hfYFP structure, the...

...(you generally don’t want more tag than protein), 2-3 tags is increasingly common. Tandem affinity purification...TEV cleavage site (more on that in a moment), and 2 ProtA IgG-binding domains. TAP has since come to encompass...found in this paper (available at Addgene).   Table 2: Protease recognition sites commonly used with tags...for your protein. If you’re working with an uncharacterized protein, or a protein for which a good antibody...

...preexisting resistant subpopulation 2. Resistant cells among the separate replicates would have different ...medicine 21.5 (2015): 440-448. Pubmed PMID: 25849130. 2. Hata, Aaron N., et al. "Tumor cells can follow distinct... population using the library, propagated it, separated it into roughly identical replicate groups, and...depiction below): 1. Resistant cells among the separate replicates would contain the same labels - i.e...approach is in contrast to the current treatment paradigm where resistant populations are treated as they...

...vector:insert ratio is used in the DpnI digestion and 2 µL of digest product are used in the transformation...Biology 38 (2016): 145-154. PubMed PMID: 27391134. 2. Li, Chaokun, et al. "FastCloning: a highly simplified...FastCloning, you PCR both the vector and the insert in separate reactions and then combine the products and digest...

...recognize Fab fragments from the same species and 2) these fragments will be more likely to bind (and ...epitopes recognized by the secondary.   Figure 2: There are a few common solutions to avoid background...biorender.com. Proper tissue processing and sample preparation can also help reduce the levels of endogenous...

...Epub 2016 May 2. PubMed PMID: 27136078. Find plasmids from this publication at Addgene. 2. Swarts, Daan...against invading DNA. In 2014, Swarts et al. characterized Argonaute from Thermus thermophilus (TtAgo),...

...PMCID: PMC3785459. 2. Krashes MJ, Koda S, Ye C, Rogan SC, Adams AC, Cusher DS, Maratos-Flier E, Roth BL,... tropism and axonal transport of AAV serotypes 1, 2, 5, 6, 8 and 9 in the mouse brain. PLoS One. 2013 ...individual gene knockouts (using gRNA lentiviruses). Separating Cas9 and the gRNA into two constructs (i.e., ...between experiments and over time (as compared to separate transductions with a single-vector system). Tips...

... Genes Dev. 1988 Jun;2(6):718-29. doi: 10.1101/gad.2.6.718. PMID: 2843425.Takara Bio. Tet-On and Tet-Off...Targeted Gene Expression in Drosophila.” Genetics 186(2) (2010): 735-55. PubMed PMID: 20697123. PubMed Central... in Saccharomyces cerevisiae.” FEMS Yeast Res. 12(2) (2012): 197-214. PubMed PMID: 22129153. Additional... the system have been engineered and improved. Takara’s “2nd generation Tet-Off vectors (Tet-Advanced)...this way, you can interrogate the activity of uncharacterized promoters. Similarly, placing UAS upstream ...-Off systems: second generation. https://www.takarabio.com/products/gene-function/tet-inducible-expression-systems...

... pCRISPomyces-2 contains Cas9 and a gRNA cassette. The simpler system of pCRISPomyces-2 displays a higher...and a subsequent curing protocol that requires only 2-3 hours incubation. E. coli and T. citrea scarless...four major components including (1) a CRISPR RNA, (2) four proteins forming the QCascade DNA-targeting ...expresses lambda Red and Cas9, which are separately inducible by L-arabinose and anhydrotetracycline (aTet), respectively... template or non-template strand, among other parameters. .  Stanley Qi lab's CRISPRi plasmids Stanley...across multiple gRNAs per gene (Singh et al. 2016). Sarah Fortune's lab M. tuberculosis CRISPRi plasmids Noting...proteotoxic when used in Mycobacterial species, Sarah Fortune’s lab screened 11 other Cas9 orthologs to...

...nonsense-mediated decay. Although Nelson et al. observed only 2% genome editing in one experiment, they found the ... cells throughout the body, especially stem cells 2. Remove any immunogenicity of the AAV vector Once ...Science. 2015 Dec 31. doi:10.1126/science.aae0169 2. Long C, Amoasii L, Mireault AA, McAnally JR, Li H...highest-ranking predicted off-target sites. Each paper characterizes additional, unique facets of CRISPR DMD therapy...effectiveness and off-target risk must be evaluated separately, increasing the time to clinical approval. Even...Blog Learn How CRISPR Could Be Used to Treat Cataracts and Cystic Fibrosis Read How Homology Directed...

...wisdom  Reach out to 2-3 individuals in your professional circle to vouch for the character you painted in ...and scholarships in different subject areas!    2. Resume and cover letter know-how  Tailor your resume...increase your competitive edge, they alone, are no guarantee of success. Don’t luck into an internship, prepare...

...episode 2 "Organizing Literature" : I found out about Zotero here Dear Grad Student podcast episode 2 "Essential...flagging passive voice, overuse of adverbs, and paragraphs with many hard-to-read sentences.  Grammarly ...

...linear fashion. This implies that reducing the power 2-fold does not reduce photobleaching by half. How exactly...prolonged time). Other illumination strategies, such as 2-photon-excitation, selective plane illumination, TIRF...al, 2017) and cannot be described by a single parameter. Therefore, it is necessary to know how the fluorescence... power, the photostability of the variants is comparable (lower panel of figure 1). To avoid changes in...microscopy (University of Amsterdam). He develops, characterizes and uses genetically encoded fluorescent probes...

...studies have shown that the conditions Sotos-type 2 and Marshall Smith Syndrome are due to mutations in...accurate diagnosis of NFIX-related syndromes (Sotos-type 2 and Marshall-Smith) and could be expanded to NFIA-...manifestations. PMM2-CDG is the most frequent CDG type characterized by deficient phosphomannomutase (PMM) activity...suffer from immunological defects. These are characterized by a predisposition to severe and recurrent ... in the NFIX gene. These diseases are characterized by overgrowth phenotypes and neural developmental ...

...   1)   “Piled Higher and Deeper” by Jorge Cham   2)   “Boxplot” by Maki Naro   3)   “Bird and Moon” by...Unearthed Comics” by Sara Zimmerman   5)   “Beatrice the Biologist” by Katie McKissick       Sara Zimmerman, Unearthed...

...reach out to the corresponding potential depositors. 2. Tweet at us! Tweet @Addgene with #suggestaplasmid...where we can find the plasmid. While we cannot guarantee that everyone we reach out to will end up depositing...

..., and the desired gene (transfer vector plasmid), 2) the HIV-1 genome containing all viral genes with ...protocols that have been described (protocol 1, protocol 2, protocol 3) to find the one that will give you the...PubMed PMID: 3785217. PubMed Central PMCID: PMC367857. 2. Naldini, Luigi, et al. "In vivo gene delivery and...safer system. Unlike the MoMLV, lentiviruses, a separate genus of the Retroviridae family including human...facilitates nuclear export) is expressed from a separate plasmid, and the promoter of the 5’LTR has been... In this case your gene and the marker can be separated by an internal ribosome entry site (IRES) and ...

...residues, which can be found throughout the antibody. 2) Endogenous cysteines, such as those that make up ...Detecting antigens by quantitative immuno-PCR. Nat Protoc 2:1918–1930. https://doi.org/10.1038/nprot.2007.267 ...commercially conjugated antibodies available, that never guarantees the antibody you need will be conjugated, or ...//doi.org/10.3390/antib4030197  Hegazy M, Cohen-Barak E, Koetsier JL, et al (2020) Proximity Ligation ...

...ITR, which can either be “flip” or “flop” (Figure 2). Additionally, the ITR contains a 4-nucleotide Rep...site for Rep proteins (Savvy et al. 2013). Figure 2: AAV2 wild type ITR in flip and flop orientation. ..., B-B’, and C-C’) that give the feature its characteristic T-shape. The arrangement of the B-B’ and C-...

...misplacing your results or losing that lab notebook from 2 years ago. The free subscription to Lab Archives allots...notebooks you’re saving trees! The Earth thanks you. 2. Gibco Cell Culture by Gibco Whenever I start to learn...MSDS for a lab chemical of choice. Now, I can’t guarantee every reagent in your chemical closet is in this...

...days before the deadline isn’t going to be much use. 2. Originality is essential for grant success The two... it’s important not to throw in the towel if 1 or 2 funding bodies say no. If possible, get feedback from...perfectly reasonable paragraph unintelligible to a lay audience. My lay paragraph typically includes a...kindly friends and family to read my non-expert paragraph and point out words or sentences that are unclear...

... reduce the background staining (Table 2).      Table 2: Secondary antibody selections for a four-antibody...with users trying to probe multiple targets in parallel. In these cases, it is critical that users choose... experiment.  When probing multiple targets in parallel, use the most narrowly specific secondary antibodies... four-antibody panel, staining in parallel    In short, when staining multiple targets, look for primary...

... signal back again. The Ting lab constructed 2 versions of this reporter to target the K9 (pcDNA3-K9 ... 525.7570 (2015): 533-537. PubMed PMID: 26352475. 2. Bygren, Lars O., et al. "Change in paternal grandmothers...disrupts normal function. Angelman syndrome is characterized by intellectual and developmental disability...or smiling, whereas Prader–Willi syndrome is characterized by low muscle tone, short stature, cognitive... in the FRET signal. Subsequent demethylation separates the fluorescent units and so lowers the FRET signal...

...represent a favorable state for tumor progression (Ref 2). Future treatments may need to target this immunosuppressive...cell 30.1 (2016): 147-160. PubMed PMID: 27411589. 2. Todoric, Jelena, Laura Antonucci, and Michael Karin...the Relationship” by our guest blogger Subhadra Jayaraman, a doctoral candidate at Binghamton University...

...populations that may otherwise be difficult to separate out. In Figure 2, you can see that, without the FMO, it... from the Ly6C positive population. Figure 2: An FMO gating example. The FMO sample (A) can be ...

... Zetsche et al., Nat Biotechnol. 2015 Feb 2;33(2):139-42. doi: 10.1038/nbt.3149. Light-inducible ...and on your favourite signalling pathways (Figure 2). They are all available now at Addgene. 1 Lungu...20;19(4):507-17. 10.1016/j.chembiol.2012.02.006. 2 Guntas et al., Proc Natl Acad Sci USA 2015 Jan 6; ...Nihongaki et al., Chemistry and Biology. 2015 Feb 19; 22(2):169-74. High efficiency cloning for enzyme screening...inducible, gene knockdown with shRNA or shRNA-miR30, (2) tamoxifen-inducible CreERT2 gene deletion, (3) simultaneous...different fluorescent proteins, 8 constitutive promoters, 2 includible promoters, 3 polyA terminators and various...of plasmids required for each input/output system; 2) removing all chemically inducible promoters; 3) improving...

...knock-out cell line to perform downstream characterization of, isolating 2 or 3 distinct clones to perform key...sites would confound your experimental results and 2) perform targeted off-target measurements at those...experiments where only localization of the Cas occurs. 2 gRNA approach Off-targets that come from mutations...

...somewhere else (Fig. 2). Class II TEs have LTRs on both of their ends.    Figure 2: Overview of DNA...has four major components: 1) a CRISPR RNA (crRNA), 2) four proteins (TniQ, Cas8, Cas7, Cas6) that forms...another. This jumping is what caught the eye of Barabara McClinntock who first discovered transposons due...

...detection, and control. Int J Pharm. 2018 Oct 25;550(1-2):251-268. doi: 10.1016/j.ijpharm.2018.08.043. Epub...and particle formation. J Pharm Sci. 2015 Feb;104(2):602-11. doi: 10.1002/jps.24259. Epub 2014 Nov 21....Roberts, Prasad S. Sarangapani, Protein aggregation, particle formation, characterization & rheology, Current...are contained to one plasmid or if they’ve been separated onto two. You’ll first transfect your cells with...harvesting recombinant antibodies consists of separating antibodies from media, cells, and debris. Affinity...bind ligands on the beads and a magnet is used to separate the beads from the media (Brechmann et al. al.... of your prep. In this method, antibodies are separated by size on an SDS-PAGE gel. Once stained, the ...

...Hrit et al. 2018, Kaas et al. 2013). Figure 2: CRISPR TET-based epigenetic editing. In reality, ...approach for targeted DNA demethylation. Cell Discov 2, 16009. https://doi.org/10.1038/celldisc.2016.9.  ...hear it (whether at a scientific conference or a karaoke bar), you probably want to sigh. The problem is... directly inside the gRNA sequence is a great guarantee that DNMT1 will be blocked (within PAM works, ...

...amilCP, tsPurple, and gfasPurple) (Figure 2). Figure 2: Chromoprotein expression from bacteria on.... https://doi.org/10.1007/bf00428249 Chan MCY, Karasawa S, Mizuno H, Bosanac I, Ho D, Privé GG, Miyawaki...Miyawaki A, Ikura M (2006) Structural Characterization of a Blue Chromoprotein and Its Yellow Mutant from ...Hoegh-Guldberg O (1995) Isolation and Partial Characterization of the Pink and Blue Pigments of Pocilloporid...

...Indicators GEVIs can be divided up structurally into 2 main groups: voltage sensitive domains and Rhodopsin...depending on your experimental system.   Figure 2: General overview of chemigenetic tools. A genetically...mechanism of their voltage sensing component, which separates them into two distinct categories detailed below...phosphatases from sea squirts. This design structurally separates the two components of the indicator, which can...FP-rhodopsin GEVIs have been engineered to have kinetics comparable to its one component counterpart and is significantly...