...released from the cell like baby spiders from an egg sac. (Okay, some enveloped viruses bud out, instead of...

...phenotype (using sacB, Figure 1, further discussed below).  Counter-selectable marker: sacB What are the...system relies on the sacB gene product of B. subtilis (Hmelo et al., 2015). The sacB gene product is a sucrase...contain sacB and will not grow on sucrose. One popular counterselection vector is the pDS132 sacB-based...have undergone a double crossover will not contain sacB, allowing them to grow on sucrose. Cells that have...sacB-based allelic exchange vector. This vector contains sacB, an chloramphenicol resistance cassette, and the...acquire the ability to grow on sucrose, perhaps by sacB inactivation.  Bacterial toxins for counter-selection...counter-selection than one merely co-opted toward that aim (i.e. sacB). Importantly, to prevent activation of toxins before...

...interest. SacB counter-selection SacB counter-selection relies on the toxic product produced by the SacB gene...that contain constructs that have lost the sacB gene. SacB can be used similarly to CcdB. For example,...lab, the sacB gene is used for negative selection on 5% sucrose plates. Exchange of the sacB gene for ...gene. sacB comes from the gram-positive bacteria Bacillus subtilis and encodes the enzyme levansucrase ...antibiotic resistance genes, toxin genes, and the sacB gene). By tapping into nature’s toolbox, scientists...

...aureus Cas9 (SaCas9, 1053 amino acids) displayed cleavage activity in mammalian cells. SaCas9 produced indels...vivo studies. Testing AAV-SaCas9 in vivo To test the efficiency of AAV-SaCas9 in vivo, the Zhang lab created...SpCas9, leading the group to focus their efforts on SaCas9 characterization for in vivo studies. One of the...To compare the off-target effects of SpCas9 and SaCas9, Zhang’s group used an approach called BLESS (direct...). Using this sensitive method, they found that SaCas9 did not display higher levels of off-target activity...created an all-in-one SaCas9 and sgRNA construct using the liver-specific serotype AAV8. Since the efficiency...compared to a control AAV-GFP. Not only did the AAV-SaCas9-sgRNA constructs mediate genome modification, but...

...Many thanks to our guest blogger Jessica Sacher! Jessica Sacher is a microbiology PhD student at the University... post was contributed by guest blogger, Jessica Sacher, a microbiology PhD student at the University of... approaches a host cell surface are complex polysaccharide structures, many of which are easily varied...description of a technique published by Simpson, Sacher, and Szymanski in January 2016 in Viruses (5). ...what her cat is thinking. Find her on Twitter @JessicaSacher.   References 1. Nobrega, Franklin L., et al...PMCID: PMC4971087. 3. Simpson, David J., Jessica C. Sacher, and Christine M. Szymanski. "Exploring the interactions...PMID: 27319741. 3. Simpson, David J., Jessica C. Sacher, and Christine M. Szymanski. "Development of an...

...’s very sad because it’s years of work,” Sacher says. Sacher hopes that as Phage Directory expands, it...When Jessica Sacher, a microbiologist from the University of Alberta, saw that scientists were using ...really needs it, but then it might to be too late,” Sacher says. As the labs in the directory step forward...Since the inception of Phage Directory ~1 year ago, Sacher and Zheng found that they could expand their role...information and potential for successful phage therapy, Sacher and Zheng also saw the need to archive phage reagents...

...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...

... Bujard created the tetracycline-controlled transactivator (tTA). They fused TetR with the transcriptional...by Gossen and Bujard, both the promoter and transactivator components of the system have been engineered... in a similar manner to the Tet system. The transactivator configuration of the system is a positive repressible...the cmt and cym bacterial operons. A chimeric transactivator, cTA, was created by fusing CymR to the activation.... Riabinina et al. also developed chimeric transactivators GALQF and LexAQF, which activate their respective...genes for metabolic engineering applications in Saccharomyces cerevisiae.” FEMS Yeast Res. 12(2) (2012): 197...

...from Zygosaccharomyces rouxii), B2 (from Zygosaccharomyces bailii) and B3 (from Zygosaccharomyces bisporus...as low toxicity. FLP/FRT recombinases (from Saccharomyces cerevisiae) had been used successfully in Drosophila...

...ultracentrifugation altogether. Chomczynski and Sacchi showed that RNA could be effectively separated ...method are still widely used today (Chomcynski and Sacchi, 2006).  View our protocol for RNA extraction! ...https://doi.org/10.1021/bi00591a005 Chomczynski P, Sacchi N (1987) Single-Step Method of RNA Isolation by...doi.org/10.1016/0003-2697(87)90021-2 Chomczynski P, Sacchi N (2006) The single-step method of RNA isolation...

...their production does require continued animal sacrifice. Could there be an alternative method for immunodetection... which means their production requires animal sacrifice and precludes genetic engineering of a tag or ...available Not recombinant; Require animal sacrifice Poor tissue penetration Less resolution due to...Nanobodies   Recombinant; do not require animal sacrifice Good tissue penetration Can be labeled with multiple...

... SpCas9-VQR TadA-TadA* NGA 8 SaCas9 TadA-TadA* NNGRRT 9 SaCas9-KKH TadA-TadA* NNNRRT 8 SpCas9-...the SpCas9 with the mutant versions of SpCas9 and SaCas9, which recognize PAM sequences other than NGG (...Target-AID (PmCDA1) NGG 5 SpCas9-VQR rAPOBEC1 NGA 8 SaCas9-KKH rAPOBEC1 NNNRRT 8 ABE SpCas9 TadA-TadA* NGG...

...trigger spontaneous swimming  Researchers at the Isacoff lab applied optogenetics in zebrafish larvae to... activated with light.  Find plasmids from the Isacoff lab! They then tested what kind of swimming behavior...versatility of the zebrafish model organism, the Isacoff lab was able to link these cells to their pivotal...

...site, SacI restriction site and a linker region. The PCR product is then ligated into the MCS/SacI site... which will ultimately co-localize the VP64 transactivator with a dCas9 that has been targeted to a very...genome Thoru Pederson’s lab at the University of Massachusetts Medical School has recently developed a CRISPR-based...Lab at the Wyss Institute have extended the transactivating capabilities of CRISPR/dCas9-fusion proteins...proteins and have made their dCas9 transactivators available through Addgene. The development of dCas9 and ...

...post. Fatima Enam: Biosensors for human milk oligosaccharides Fatima Enam is currently a graduate student...fluorescent protein-based biosensors for Human Milk Oligosaccharides (HMOs). HMOs are thought to play an important...Resources on the Addgene Blog Learn More about Photosactivatable Fluorescent Proteins Use Light to Control ...

...Tales of Music and the Brain by Oliver Sacks "Neurologist Oliver Sacks examines the relationship between the...therapeutic properties it has. As with other works of Dr. Sacks, Musicophilia includes case studies of relevant ...

...this problem, the Zhang lab has previously used SaCas9 (1053 aa/3.2 kb) with AAV vectors for efficient...and 3.9 kb in length, respectively, larger than SaCas9 but smaller than SpCas9. These nucleases should...that of the widely used SpCas9 and comparable to SaCas9. As seen with Cas9 orthologs, Cpf1 efficiency varies...

... thanks for our guest blogger, Gairik Sachdeva! Gairik Sachdeva is a Ph.D. in Bioengineering and works...This post was contributed by guest blogger Gairik Sachdeva. Healthcare consulting is a fast-paced field, ...

.... For example, Cas9 from Staphylococcus aureus (SaCas9) is about 1 kilobase smaller than SpCas9, so it...Campylobacter jejuni (CjCas9) is even smaller than SaCas9 and is also compatible with AAV delivery. NmCas9...Addgene Blog  Read more about Cpf1 Read more about SaCas9 and AAV Check out our CRISPR Featured Topic Page...

...non-temperature-sensitive replicon, and 3) added the sacB gene for counterselection to yield pEcCas. In pTargetF...growth of its E. coli host, and the addition of a sacB gene renders it beneficial for plasmid clearance...in all three plasmids. pCutamp is then removed by sacB counterselection.  The MUCICAT toolkit includes...