...shorter plasmid curing time. pCRISPomyces for editing in Streptomyces  Streptomyces bacteria produce a wide...created two “pCRISPomyces” systems for use in Streptomyces (Cobb et al., 2015). pCRISPomyces-1 includes ...array, while pCRISPomyces-2 contains Cas9 and a gRNA cassette. The simpler system of pCRISPomyces-2 displays...for transcriptional silencing Mycobacterium tuberculosis and Mycobacterium smegmatis. In addition to their...carries a spacer targeting the gene of interest and kanamycin resistance E. coli carrying the phage recombineering...Gibson Assembly or restriction enzyme cloning. Streptomyces bacteria are more recombinogenic than E. coli...efficiencies or can be proteotoxic when used in Mycobacterial species, Sarah Fortune’s lab screened 11 other...

... labs and hinders experiments is mycoplasma contamination. Mycoplasma contamination has been shown to ...routinely test for mycoplasma contamination. There are several commercially available mycoplasma detection kits...larger microbes such as yeast, fungi, or bacteria, mycoplasma can be extremely hard to detect with levels reaching...cells per ml before the media becomes cloudy. Mycoplasma compete with host cells for nutrients and can...the cell line’s growth and behavior. Signs of a mycoplasma contamination can be subtle and may include reduced...positive controls. Routinely test your cell line for mycoplasma and discard it if the culture has become overly...References 1. Hay, R.J., Macy, M.L. & Chen, T.R. “Mycoplasma infection of cultured cells.” Nature 339 (1989...

...subunit S12 of 30S ribosome Confers resistance to streptomycin Tn10   Confers resistance to tetracycline ...galK rpsL (StrR) endA1 nupG fhuA::IS2 DB3.1 Streptomycin  HB101 derivative. For propagating plasmids ...lacY1 proA2 rpsL20(Smr) xyl5 Δleu mtl1 DH10B Streptomycin MC1061 derivative. General cloning and storage...lacZYA-argF)U169, hsdR17(rK- mK+), λ– HB101 Streptomycin Hybrid of E. coli K12 and E. coli B (mostly ...proAB+ lacIq lacZΔM15] hsdR17(rK-mK+) JM110  Streptomycin  For storing plasmids that should not be Dam...proAB+ lacIq lacZΔM15] hsdR17(rK-mK+)  MC1061 Streptomycin Parent of DH10B/TOP10 and derived strains, common...lac-proAB) mcrA Δ(mcrBC-hsdRMS-mrr) λ-  Stbl3  Streptomycin Derived from HB101. For cloning into and storage...

...methionine synthesis (5). The budding yeast, Saccharomyces. cerevisiae, makes a great reference organism... or not proteins from the fission yeast, Schizosaccharomyces pombe, have the same function as their counterparts...yeast for budding geneticists: A primer on the Saccharomyces cerevisiae model system. Genetics 197, 33-48...yeast for young geneticists: A primer on the Schizosaccharomyces pombe model system. Genetics 201, 402-423...Surdin-Kerjan, Y. Metabolism of sulfur amino acids in Saccharomyces cerevisiae. Microbiol Mol Biol Rev 61, 503-532.... et al. Functional characterization of the Saccharomyces cerevisiae genome by gene deletion and parallel...

...where recombination has occurred. Neomycin, puromycin, and hygromycin resistance genes are commonly used...insert a resistance gene. The vector contains a neomycin resistance gene (NeoR) flanked by homology arms...remove a resistance gene. In this construct, the neomycin resistance gene is floxed so that it can be removed...the sequence of GFP is cloned together with the Neomycin resistance (NeoR) gene between exon 1 and 3 of...

...from Kluyveromyces drosophilarum), R (from Zygosaccharomyces rouxii), B2 (from Zygosaccharomyces bailii...as low toxicity. FLP/FRT recombinases (from Saccharomyces cerevisiae) had been used successfully in Drosophila...bailii) and B3 (from Zygosaccharomyces bisporus.) Reporter constructs equivalent to site-STOP-site-myrRFP...

... ug/mL Bleomycin glycopeptide Induces DNA breaks Bactericidal 5-100 ug/mL Erythromycin macrolide...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...

...result in a spectinomycin resistant phenotype in bacteria. While there are many known spectinomycin resistance...generate bacteria resistant to both spectinomycin and streptomycin, but could also be used for studying...five dispersed regions of the target gene of spectinomycin, rpsE. These results not only uncovered novel...

... gentamicin, hygromycin, kanamycin, nourseothricin (clonNat), spectinomycin, streptomycin, tetracycline...maintain a glycerol stock. Choose from: ampicillin, apramycin, blasticidin, bleocin (zeocin), chloramphenicol...

..., and is routinely tested for mycoplasma contamination using mycoplasma detection kits. Cell line is maintained... tested for mycoplasma contamination. To date, Addgene has never had a case of mycoplasma contamination...normalization purposes are being used. Quality Control Mycoplasma Our 293T cell line was obtained directly from...

...SpCas9 is fused to the puromycin resistance gene through a 2A peptide. The puromycin resistance gene enables...causes the ribosome to translate Cas9-2A and the Puromycin resistance gene as separate peptides. PX459 This...predecessor, which had a point mutation in the puromycin resistance gene that made it less effective in...

...80S; inhibits chain elongation 100-800 ug/mL Hygromycin B hygB HeLa, NIH3T3, CHO, Jurkat T cells Blocks...80S; inhibits chain elongation. 50-500 ug/mL Puromycin pac HeLa, 293HEK, Jurkat T cells Inhibits protein...do not confuse this with G418 (aka Geneticin). Neomycin should not be used for mammalian expression – ...confers G418 resistance; however, like gentamicin, neomycin is typically used to suppress bacterial growth...

...Gluconacetobacter xylinus, Haemophilus influenzae, Hypomycrobium X, Legionella pneumophila, Methylophilus methyltrophus...calcoaceticus, Actinobacillus pleuropneumoniae, Actinomyces naeslundii, A. viscosus, Aerobacter aerogenes...Methylophilus methylotrophus, Moraxella spp., Mycobacterium aurum, M. smegmatis, Paracoccus denitrificans...spheroides, Salmonella spp., Serratia marcescens, Streptomyces lividans, Synechococcus spp., Thiobacillus ferrooxidans...

...backbone contains a kanamycin resistance cassette, you would plate the cells on kanamycin-containing agar..... Only cells carrying plasmids that contain a kanamycin resistance cassette will grow. However, often ...

...different selection markers (blasticidin, neomycin, puromycin, hygromycin) and fluorescent protein reporters ...

...marker such as puromycin, you can (also) select for the positive colonies by puromycin selection. You ...transfected cells (for example, plasmid with a puromycin resistance cassette, such as PX459). After you...

...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 ...

...but was found to have a point mutation in the puromycin cassette that makes it less efficient in some ...with customizable sgRNA from the U6 promoter and puromycin resistance from the EF-1a promoter. It’s a 3rd...PX459) V2.0. It contains a point mutation in the puromycin cassette that makes it less efficient in some ...

...Nathans, Daniel. "Puromycin inhibition of protein synthesis: incorporation of puromycin into peptide chains...Yarmolinsky, Michael B., and L. Gabriel. "Inhibition by puromycin of amino acid incorporation into protein." Proceedings...

...kinase promoter drives expression of puromycin. Puro R Puromycin resistance gene for selection of pLKO...sulfate, and puromycin are located in the “Appendix”. F.2. Determining the Optimal Puromycin Concentration...differently to puromycin selection. Addgene strongly recommends that you determine the optimal puromycin concentration...to fresh puromycin-containing media every other day. f. The minimum concentration of puromycin that results...with finer increments of puromycin to determine a more precise optimal puromycin concentration.) F.3. Protocol...expression of the puromycin resistance gene. f. To select for infected cells, add puromycin to the media at...positive control for the puromycin selection. Days 4+: g. Change to fresh puromycin-containing media as needed...