... expressing fluorescent proteins will be used to tag individual strains of bacteria. Their interactions...Understanding drug resistance mechanisms of Mycobacterium tuberculosis Dr. Patricia Sheen, Universidad...interested in understanding the mechanisms behind Mycobacterium tuberculosis resistance to pyrazinamide (PZA...methods and improved therapies for PZA-resistant Mycobacteria tuberculosis. Investigation of multispecies ...such as pACBSR-hyg and pFLP-hyg will be used to create gene deletions of important unknown regulatory ...sense contaminants and emit light as a response. To create this bacterial sensor, he will construct plasmids...

...DNA glycosylase inhibitor with a degradation tag (LVA tag) to achieve specific point mutations in E. coli...plasmid contains all of the desired gRNAs. This increases the chance that all the edits you want to make... cleave outside of their recognition sequence, creating flanking overhangs. These overhangs can be customized... ideal number. First you generate four unique kanamycin-resistant plasmids, each containing a different...construct, you clone all but one of your gRNAs into spectinomycin-resistant plasmids pX330S-2 to pX330S-(last ...Expression Systems (MuLE) Kit: This kit can be used to create lentiviral vectors expressing wt humanized Cas9...multiple promoter-gRNA cassettes, making them advantageous for small capacity vectors like AAV. In addition...

...promoters, terminators, solubility and purification tags, connectors, cDNAs, and more to find the perfect...constitutive and inducible promoters, linkers, and tags makes EcoFlex a great option for those interested... a larger library of parts but don’t need to add tags to your proteins, then the CIDAR Kit is an excellent... technology has been used to optimize protein secretion and expression in the biotech workhorse yeast ...Engineering disease resistance through gene stacking, increasing production of a protein of interest, and improving...genetic circuits with multiplexed guides, mini screening libraries, direct edit optimization, and more....can be invaluable for more complex editing and screening experiments in organisms that don’t yet have these...

...Photoactivatable GFP tagging cassettes for protein‐tracking studies in the budding yeast Saccharomyces cerevisiae...FPs) offer scientists a simple yet powerful way to tag cellular proteins and investigate protein localization...fusion, etc). For example, by engineering PA-GFP tagged to a mitochondrial matrix protein (mito-PAGFP) ... by performing several rounds of random mutagenesis screening for enhanced RFP variants. PA-mCherry (E26V...photoswitching. In 2008, scientists used mutagenesis screens to find a version of Dronpa that doesn't ...an FP-chimeric protein may be at any one of many stages of synthesis and degradation. For this reason it...photoconverts predominantly to the anionic form, causing increased fluorescence when excited with 488nm blue light...

...perfect empty backbone, made sure to add the right tags to your gene, and may have even put a fluorescent...recombination, increases plasmid stability recBCD Exonuclease V activity abolished Increased plasmid stability...mtl1 DH10B Streptomycin MC1061 derivative. General cloning and storage, blue/white screening, leucine auxotroph...) TOP10 Streptomycin MC1061 derivative. General cloning and storage, blue/white screening. F- mcrA Δ...that make major improvements such as those that increase plasmid yield and/or DNA quality. Table 1 below...sensitive. dnaJ Mutation in a chaperonin gene Increases the stability of certain expressed proteins ...methylate DNA lac Lac operon mutations Blue/white screening of clones   lacIq lac repressor overproduced...

... al. shows just that - using MMEJ to insert FLAG tags into the genome of the pathogenic fungus Aspergillus...of the gene it follows. For increased versatility, it would be advantageous to adapt PITCh to insert genes... deletions or homology-directed repair (HDR) to create precise genome edits. But have you heard of another...depositor Takashi Yamamoto’s lab has harnessed MMEJ to create a new method for CRISPR gene knock-in, termed PITCh...PITCh plasmids, GFP knock-in cell lines can be created in about a month and a half, without the need for...of press. However, this process accounts for a percentage of the mutations seen with TALENs and CRISPR.... between the microhomologies.MMEJ has certain advantages over HDR. Some species don’t have good HDR systems...

...chapters on genome engineering, cloning, and plasmid tags, addtional content added to the "Plasmids that Glow...resistant plasmid, pSC101, and a newly developed kanamycin resistant plasmid, pSC102, with EcoRI and selected...Plasmids 101 eBook! In keeping with our goal of creating a one-stop reference guide for plasmids we've ...

...uracil DNA glycosylase inhibitor and degradation tag on the fusion protein to limit the activity of the... lab created two “pCRISPomyces” systems for use in Streptomyces (Cobb et al., 2015). pCRISPomyces-1 includes...other advantages including shorter plasmid curing time. pCRISPomyces for editing in Streptomyces  Streptomyces...array, while pCRISPomyces-2 contains Cas9 and a gRNA cassette. The simpler system of pCRISPomyces-2 displays...proteotoxic when used in Mycobacterial species, Sarah Fortune’s lab screened 11 other Cas9 orthologs to...for transcriptional silencing Mycobacterium tuberculosis and Mycobacterium smegmatis. In addition to their...Addgene depositor Luciano Marraffini’s lab took advantage of this lethality to design the first synthetic...

...the concentration of inducer or adding an affinity tag such as GST may help with solubility issues. We ...toxic or insoluble proteins Origami2 (DE3)** Streptomycin and Tetracycline Contains highly active thioredoxin...glutathione reductase to faciliate proper folding; may increase multimer formation Δ(ara-leu)7697 ΔlacX74 ΔphoA... General protein expression  m15 pREP4*, ** Kanamycin (pREP4) Cis-repression of the E. coli T5 promoter...salts may be preferable if the protein product is secreted to the medium or if slow expression is required...required due to solubility concerns. Insoluble and Secreted Proteins: The most common purification protocols...

...developing zebrafish pancreas. They first tagged these cells using plasmids. Then using HOTcre, a method that...of quiescent and proliferative pancreatic β-cells identified by HOTcre mediated labeling." Proceedings...researchers: the mouse (Mus musculus). Mice have many advantages as a mammalian model organism for scientists ...You can find these plasmids at Addgene. Another advantage of mice as model organisms is that they are genetically...in the fly (Pandey and Nichols, 2011). A huge advantage of using the fruit fly is the array of genetic...these systems check out Gerald Rubin’s lab who created a set of modular vectors that can be used to optimally... of the fly (Pfeiffer et al., 2010). Yeast (Saccharomyces cerevisiae) Yeast, one of the simplest eukaryotic...

...Addgene is certainly going to have to find ways to tag and curate reagents from these emerging organisms... progress on the NSF “G to P grand challenge.” Creating a productive, new scientific community is no small...in particular, that they all share the plasmids created under this programs via Addgene. New organisms ...So, think beyond Drosophila melanogaster or Saccharomyces cerevisiae (both fine research organisms) -  ...-  perhaps you’ll choose to study Crepidula atrasolea or Prochlorococcus with your next project. Gymnotiformes...crisis. I like how they are using protocols.io to create a protocol community! Asclepias syriaca and Asclepias...used to study aspects of cognition. Spiralians - Crepidula atrasolea (Jonathan Henry) This black footed slipper...

...LDB) to the C-terminus of FLP or Cre (pCAG-CreERT2 #14797). These Cre/ FLP versions are retained in the...control without modification - either Cre is there or it’s not. Cre-mediated possibilities for site specific...control FLP and Cre expression have been developed. Read on to learn more about FLP-FRT, Cre-lox, and how...the control of Cre recombinase are used in combination with mouse lines that express Cre only in the cell...Tamoxifen inducible Cre versions can be used in these experiments. This elegant combination of Cre, FLP, and inducible...about Cre-Lox Check out Retrograde AAVs for Neurscience Research Resources on Addgene.org Find Cre-Lox...contributed by guest blogger Katrin Michel. Cre-lox is an incredibly popular and powerful site specific recombinase...

...Slow transformation   Spectinomycin/streptomycin Spectinomycin and streptomycin both inhibit protein synthesis.... Pros: Stable (spectinomycin). Cons: Cost (spectinomycin) or stability (streptomycin), doesn’t work for... for transformations. Con: Expensive.   Kanamycin Kanamycin, or kan, is an aminoglycoside which inhibits...Like carb, spectinomycin is the more stable and expensive antibiotic compared to streptomycin. Spectinomycin...cleavage in human cells. Mutagenesis, 20(2), 111–114. https://doi.org/10.1093/mutage/gei016I Resources on ... plates due to the fact that beta-lactamase is excreted. Satellite colonies are small colonies of bacteria...Spectinomycin/streptomycin can be successfully used for plant and bacterial culture, but some types of bacteria...

... baseline voltage), the investigators found that administering these AAVs with a dilute Cre-expressing... Ace2N-mNeon-A122D and Voltron2 are available as Cre-dependent AAV1 vectors and are suitable for extended...CRISPR screens By Emily Bentley Building on their CROPseq method for optical pooled CRISPR screening, Paul...pooled screens in human cells. Addgene offers vectors for CROPseq-multi using selection by Puromycin, Zeocin...Voltron2 voltage sensors By Brian O’Neill New viral vector preps expressing the Voltron2 voltage indicator...variant has increased signal-to-noise ratio and better ability to record subthreshold voltage fluctuations...Multiplexed perturbation and decoding in pooled CRISPR screens Improved prime editor PE7 Antibodies to facilitate...

...backbone contains a kanamycin resistance cassette, you would plate the cells on kanamycin-containing agar....positive selection, negative selection, and/or screening. We’re focusing on positive and negative selection...in this blog post, but don’t worry, we’ll cover screens in a future post. Positive selection: Cells that.... Only cells carrying plasmids that contain a kanamycin resistance cassette will grow. However, often ...with or without your inserted DNA) and further screening will help you determine if the plasmid backbone...successful uptake of your DNA of interest by taking advantage of bacterial characteristics such as prototrophy...arises in yeast. For example, Markus Ralser’s lab created a yeast prototrophy kit containing 23 plasmids ...

... ug/mL Bleomycin glycopeptide Induces DNA breaks Bactericidal 5-100 ug/mL Erythromycin macrolide...Additionally, the presence of a plasmid is disadvantageous from the bacterium's perspective – a plasmid-containing...Class Mode of Action*   Working Concentration** Kanamycin aminoglycoside Binds 30S ribosomal subunit; ...mis-translation Bactericidal 50-100 ug/mL Spectinomycin aminoglycoside Binds 30S ribosomal subunit;...protein synthesis Bactericidal 7.5-50 ug/mL Streptomycin aminoglycoside Inhibits initiation of protein...Plasmids 101 Posts Learn How to Use Blue White Screening to Simplify Your Cloning Experiments See Where...

...different selection markers (blasticidin, neomycin, puromycin, hygromycin) and fluorescent protein reporters ... both of these cases, transposons combine the advantages of viruses and naked DNA while eliminating some... then degraded by the cell.4.) The transposase creates a double strand break in the DNA and allows the...transposase gains its name from the fact that it was recreated from a defective transposase found in fish. This...transposases. Improvements have been made since then by increasing the consensus between a greater number of extinct...primary cells. In 2009, Zsuzsanna Izsvak’s lab screened many SB tranposase variants for hyperactivity ...

...yet they still share many of the same technical advantages that come with working with prokaryotes. This...primarily pertains to ORIs in budding yeast, Saccharomyces cerevisiae; however, we’ve also noted some features...required for the replication of fission yeast, Schizosaccharomyces pombe, vectors at the end.) In a previous...must be utilized or a new combination needs to be created in advance of the experiment. The marker provided...physiological levels due to high copy numbers. This creates a potential metabolic burden on the yeast cells...

...contains 24 copies of SunTag, a synthetic scaffold that can recruit GFP fused to the SunTag-specific antibody...Imaging by Coat protein Knock-off) instead takes advantage of the ribosome’s movement. When the ribosome ...Using translation inhibitors cycloheximide and puromycin, Halstead et al. verified that translation is ...indicating translational repression. During later stages, single RFP-labeled osk and Oskar protein were ...scFv. Each time the reporter is translated, the SunTag sites on the nascent polypeptide recruit scFV-GFP...translationally silent. Both of these methods increase our understanding of translation dynamics, but...but they come with a few caveats. In both cases, creating a reporter mRNA requires inserting multiple hairpin...

...should have a fitness advantage over proteins with single PAM recognition. Created with BioRender.com...genome-wide off-targets, since increasing PAM flexibility might also increase off-target activity. Surprisingly...gone one step further, using directed evolution to create xCas9, an enzyme recognizing a broad range of PAMs...PAMs like NG, GAA, and GAT, but also displaying increased editing specificity. We’re excited to learn more...easy, right? Yes and no. If you’re just trying to create a knockout, you might be happy targeting anywhere...precise edits or knock-ins. Editing efficiency decreases drastically once your cut site is more than 10...insertion site. Base editing, another method to create point mutations, also has a strict editing window...