...Brown W, Bardhan A, Delaney M, Ilk AS, Rauen RR, Kahn SI, Tsang M, Deiters A (2020) Spatiotemporal Control...protein combination. The optimized LACE system consisted of: 1) CIBN-dCas9-CIBN, where CIBN is the N-terminal...(Kawano et al., 2015). Nicknamed paCas9-1 and consisting of the fusion proteins N713-pMag and nMagHigh1...Michael Lin, who developed a chimeric protein consisting of Cas9 and a photo-dissociable dimeric green...

...without a resistance gene, allowing resistant bacteria to multiply, spreading their resistance genes throughout...The crisis of antibiotic resistance is upon us, and the world is unprepared. Each year in the United ...antibiotic resistant bacteria. Even when researchers develop new antibiotics, the onset of resistance is swift...antibiotic resistant bacteria with a weapon that they’ll have less potential to become resistant to? CRISPR...antibiotic resistance Most antibiotics target one of three essential processes: cell wall synthesis, DNA replication...or antibiotic resistant bacteria, and inappropriate antibiotic usage selects for resistant bacteria, allowing...propagate their resistance genes. Our society is hoping to combat antibiotic resistance using multiple ...

...example, we examined ICE analysis of Sanger sequencing data alongside the analysis of NGS-based amplicon ...scalability, the ICE software has two analysis formats: sample by sample analysis, which can compare up to five...synthego.com. Overview of ICE editing analysis Once the analysis is complete, a new screen appears with...also download the entire analysis as a ZIP file by clicking the Download Analysis Data button on the bottom...bottom right of the analysis screen. ICE analysis details Contributions   The Contributions tab shows the...tool was initially created to support the CRISPR analysis needs of Synthego’s scientists, and it’s now free...sequencing data and found that the accuracy of ICE analysis results was highly comparable (within r2=0.96)...

...   Statistical analysis  Getting your RQ value isn’t the end of your analysis process. You’ll still...different statistical analysis methods that pair well with slightly modified CT analysis, so pick one and ...chemistries, primer optimization, controls, and analysis.  As a rule of thumb, you can assume that qPCR...chemistry  The two most common qPCR chemistries are hydrolysis probes (which you probably know as TaqMan) and...multiplexed Single amplicon validation technique Hydrolysis Probes (Taqman) Low; can be reduced with a ... Yes, one Yes Yes Agarose gel or uMelt analysis dsDNA dye (Sybr)  High No No No ... Yes, two Yes Yes Agarose gel or uMelt analysis Hairpin probes Low Yes, one Yes Yes...

...cancer development, metastasis, and drug resistance, Bhang says “the [drug] resistance story panned out ...cells that represent the preexisting resistant subpopulation 2. Resistant cells among the separate replicates...found that similar barcodes consistently made up the majority of the resistant populations. In fact, 88%...apply [it to].” Using ClonTracer to study drug resistance Although Bhang and co-workers started using the...determine whether or not cancer cell populations resistant to drug treatments arise during treatment or are... outcomes (see schematic depiction below): 1. Resistant cells among the separate replicates would contain...abundance before and after a treatment. The library consists of a pool of lentiviral vectors with roughly 73...

...is as a biomedical model organism.  Arabidopsis thaliana Arabidopsis thaliana is one of, if not the most...manipulation and mutagenesis techniques, are available to the scientific community.  The Arabidopsis genome project...beginning of widespread Arabidopsis genetics research development. Arabidopsis thaliana was fully sequenced...for use in CRISPR and Tol2 transposase induced transgenesis.  Xenopus Xenopus, or frogs, are one of the ...understand human disease alleles during and after embryogenesis. Addgene has over 600 plasmids that contain ...characterized genome, and widespread utilization, Arabidopsis has made significant contributions to understanding...understanding plant and crop science alike.  Arabidopsis has many of the traits that make an ideal model organism...

... designed to insert a resistance gene. The vector contains a neomycin resistance gene (NeoR) flanked by...insert and later remove a resistance gene. In this construct, the neomycin resistance gene is floxed so that...into the vector on either side of an antibiotic resistance gene. To avoid selecting for cells in which the...perform a positive selection for the antibiotic resistance gene and then a counter selection for the negative...removed from the chromosome and and replaced by the resistance gene.The gene is thus disrupted, or knocked out...occurred. Neomycin, puromycin, and hygromycin resistance genes are commonly used for positive selection...positive selection marker. Since an antibiotic resistance gene can affect a cell's phenotype, researchers...

...well as you perform analysis after analysis after analysis… Treat every analysis like a wet lab experiment... my thesis project a few years ago when my advisor and I decided to do a massive RNA-seq analysis. I had...you to perform this analysis. Having a clearly stated, specific goal for each analysis will help you locate...Before you begin, briefly state the goal of the analysis, including background on other analyses that have...approach that you will take to help you plan your analysis. There is no need to go into a lot of detail –...Always, always, write a brief conclusion of your analysis, even if your conclusion is “this approach is ...ideal because…” Including a conclusion for each analysis or task will keep you from repeating your work...

...of?  A tumor’s genesis is divided into 3 phases: initiation, progression, and metastasis. The microenvironment...altered fibroblasts and promote the development and metastasis of tumors through inciting inflammation. Speaking...microenvironments affect tumor spread and drug resistance? Although we’ve seen how multiple types of cells...story. It’s essential for a tumor to spread and to resist drugs. And to do that, let’s take a look at exosomes... case, exosomes may transport miRNA from drug-resistant cells to sensitive ones. Though this process is... expression of TAK 1, an enzyme that controls apoptosis and regulates transcription. Without TKA1, cells...cells proliferate and cannot undergo apoptosis, thus multiplying uncontrollably and building up tumors....

...involved in drug resistance Wood wanted to find out which particular pathways drive resistance to specific ...relatively short amount of time, cancers become resistant to drugs, particularly targeted therapies,” said...comprehensive understanding of how cancers become resistant, an emerging theme is that they do so by activating...proliferation, survival, differentiation, and apoptosis. For each of these 17 pathways, they identified... a time, you could identify those that confer resistance to drugs,” in a rapid, straightforward, and scalable... for use in screening for pathways that drive resistance to drugs in vitro, and they’ve shown that it ...confer any phenotype of interest, not just drug resistance,” he said. “It could [be used to screen for] ...

...Updated mini-transposon vector for bacterial mutagenesis or gene targeting Victor de Lorenzo's lab...mini-Tn5 vector that can be used to generate random mutagenesis libraries or to insert heterologous genes, reporters...this vector, called pBAM1, by conducting random mutagenesis in the soil bacterium Pseudomonas putida and ...constitutively expressed, reports on the balance of biogenesis and mitochondrial degradation in vitro and in...the complexity of assembling recombination of transgenesis constructs? A new cloning system combining the...maximum of 24 elements assembled into a final transgenesis construct. Plasmids constructed using Golden...recombination template vectors, to perform multiple site mutagenesis and create complex fusion or recombination vectors...

...from Addgene consists of two plasmids: pCas9: carries Cas9 and chloramphenicol resistance pCRISPR: carries... targeting the gene of interest and kanamycin resistance E. coli carrying the phage recombineering machinery...recombineering is unsuccessful and the wild-type sequence persists, Cas9 will cleave the gene of interest, inducing...Insertion of Transposable Elements by Guide RNA-Assisted Targeting) and the Zhang lab's system called CAST... enable gRNA-directed transposition. INTEGRATE consists of four major components including (1) a CRISPR...Streptococcus pneumoniae. Robert Husson lab's M. tuberculosis CRISPRi plasmids A group from the Husson lab... used CRISPRi to study essential genes in M. tuberculosis. dCas9 and a gRNA are expressed on pRH2502 and...

... Blog Plasmids 101: Antibiotic Resistance Genes Antibiotic Resistance: An Old Solution but a New Problem...environments. The genes encoding these functions form the basis of many molecular biology tools! Figure 1...natural plasmids. Created with BioRender.com. Resistance (R) plasmids: microbial defense Genes allowing...its host. For example, cells with a penicillin resistance plasmid can grow in spite of a hostile Penicillium...fungus trying to kill them off. While antibiotic resistance-mediated selection is indispensable for cloning...cloning work, antibiotics and plasmid-encoded resistance to them are the tools of a millennia-old microbial...like colicin typically also carry the necessary resistance genes to help cells survive their own secretions...

...of plasmid multimers. Capillary Gel Electrophoresis (CGE) analysis Another technique that can detect multimers...Levene SD. Analysis of DNA topoisomers, knots, and catenanes by agarose gel electrophoresis. Methods Mol...stability study and topology analysis of plasmid DNA by capillary gel electrophoresis. BioProcess Int. 2006 ...the supercoiled plasmid monomer, and at <6 kb, consistent with the size of a supercoiled plasmid dimer.... multimers is capillary gel electrophoresis (CGE), which has been used in plasmid vaccine and gene therapy...Plasmid Topology Monitoring by Capillary Gel Electrophoresis. Curr Mol Med. 2020;20(10):798-805. doi: 10.2174... He Y, Lacher NA, Ruesch MN. Capillary gel electrophoresis with laser-induced fluorescence of plasmid ...

...many contexts within the fly including oogenesis and cytokinesis. Why name this protein after a vegetable...hedgehog exhibit abnormal segmentation during embryogenesis. Typically, regularly spaced denticles (bristles...such as cell movement/motility, development, cytokinesis, and muscle contractions (think skeletal muscle...monster, a meiotic arrest gene required for spermatogenesis One of our smaller conference rooms is named... meiotic arrest gene that is necessary for spermatogenesis. Flies that lack cookie monster are sterile...to a failure of primary spermatocytes to enter meiosis. Testis of cookie monster mutant flies are filled...White-Cooper, 2003). Why call a gene required for spermatogenesis after a Sesame Street character? The scientists...

..., making them ideal for studying vertebrate embryogenesis. To illustrate some of the reasons that zebrafish...in developing embryos are available. Additional analysis tools have been developed to help parse and visualize...1990’s, two revolutionary large-scale random mutagenesis screens produced ~1,500 zebrafish mutants that...to study development and disease. Additional mutagenesis projects have since been initiated and today ...gene deactivation tools for zebrafish Random mutagenesis screens provided a major toehold into understanding...the zebrafish is an ideal system to observe embryogenesis live. The origins, divisions, and movements ...tracing defined a high-resolution map of early embryogenesis in time and space, and was also used to precisely...

...antibiotic/herbicide resistance and fluorescent proteins. Selection using resistance genes usually involves... editing, transformation may be identified by resistance to an antibiotic. Successful editing of transformants...occurs in T0 plants (from callus) or T1 plants (Arabidopsis floral dip). In the next generation the CRISPR...planning to use it to simplify my work with an Arabidopsis mutant that can’t set seeds. I study the development...and a spontaneous step. (B) RUBY is a reporter consisting of these three enzymes expressed under the same...   Fig. 3: (Top right) A RUBY-expressing Arabidopsis seed following floral dip transformation next...without antibiotics. (Top left) Mature silique (Arabidopsis fruit) with fertilized developing seeds from ...

...of a homologous donor (e.g., sister chromatid, plasmid, etc.), and consists of three pathways: classical...initiates primary synthesis from a template followed by a round of lagging strand synthesis to fill in the...for strand invasion and a primer for DNA repair synthesis. The ssDNA strand then displaces one strand of...classical double-strand break repair (DSBR), synthesis-dependent strand-annealing (SDSA), and break-induced ... template to serve as a primer for DNA repair synthesis, ultimately leading to the formation of double...manner, this results in a non-crossover event.  Synthesis-dependent strand-annealing (SDSA) pathway SDSA... chromosomal aberrations. Pfeiffer P, et al. Mutagenesis (2000). PubMed. Multiple Pathways of Recombination...

...eliminating stringent factor Allows RNA synthesis in absence of protein synthesis Ptrc-ccdA   Propagation of ccdB-containing...of 30S ribosome Confers resistance to streptomycin Tn10   Confers resistance to tetracycline Additionally...uptake, iron uptake receptor mutation. T1/T5 Phage resistance gal Mutation in galactose metabolism pathway...only gyrA, gyrA96 DNA gyrase mutation Confers resistance to nalidixic acid  hsdRMS hsdR(rk-, mk+) Unmethylated...constitutive expression of genes for deoxyribose synthesis Allows uptake of large plasmids hee   High electroporation...source for growth proAB mutation in proline biosynthesis pathway Requires exogenous proline source for...Table 2: Lab strains of E. coli Strain Natural resistance Primary Use Genotype ccdB Survival 2 T1R   ...

...the forefront in the so-called “reproducibility crisis.” Results in preclinical, biomedical and psychological...be able to look under the hood at the raw data, analysis code, and other materials. The use of reporting...collected, what the hypotheses are, and how each hypothesis will be tested with a specific statistical test...Preregistration makes clear the distinction between hypothesis testing work, in which a-priori hypotheses are...are tested with a new experiment; and hypothesis generation, in which data are investigated to look for...unexpected trends or differences between groups. Hypothesis generation is critical for advancement, but we...too often encouraged to present that work using hypothesis testing statistical tools that generate a p-value...