...insertions, deletions, and point mutagenesis to complex assembly of multiple genes. Primer design for each type... to the 5’ end of the reverse primer. Mutagenesis - Mutagenesis is performed by designing primers astride...included in a single PCR, for example for subcloning genes from different templates. All PCRs are run as an...@jakefwatson and @JGarciaNafria.  References 1. Jones, Douglas H., and Bruce H. Howard. "A rapid method...method for recombination and site-specific mutagenesis by placing homologous ends on DNA using polymerase...

...bacteria to multiply, spreading their resistance genes throughout the bacterial population. But what if...them to multiply and propagate their resistance genes. Our society is hoping to combat antibiotic resistance.... Above all, we need new antimicrobials, ideally ones for which the development of resistance will be .... targeted beta-lactam and quinolone resistance genes in E. coli using plasmid and phagemid delivery systems...therapy. Another strategy for CRISPR antimicrobials combines them with both replication-competent phages and...work targeted two distinct beta-lactam resistance genes, again with very high specificity. This double phage...prevents sensitive bacteria from acquiring resistance genes. Yosef et al. suggest that it could be valuable ...

...Yang, a professor in synthetic biology from the Chinese Academy of Sciences. In the Yang lab, we’re continually... consists of one plasmid that expresses the S. pyogenes Cas9, a temperature-sensitive replicon for self-curing...Fortunately, we were able to successfully delete genes of interest in BL21(DE3) using the newly generated...with multiple terminators can serve to interrupt genes (or gene clusters) in metabolic engineering. So ...a strain library with 1-11 copies of integrated genes. We obtained a stable and high-yielding strain which...guest bloggers! Qi Li obtained her Ph.D. from the Chinese Academy of Sciences (CAS) Center for Excellence...Ph.D. from Shanghai Institute of Biochemistry, Chinese Academy of Sciences. He joined Shanghai Institutes...

...sequence of interest can lead to poor expression of genes within that sequence and should therefore be prevented... recombinase are used in combination with mouse lines that express Cre only in the cell type of interest...and expression of a separate gene. In these mouse lines, gene knockout can be induced by Cre recombinase...researcher exquisite control over expression of multiple genes in specific cells at specific times. Many thanks...at MIT and is fascinated by the question of how genes and proteins coordinate billions of neurons to form...Properties of FLP Recombinase Evolved by Cycling Mutagenesis.” Nature biotechnology 16(7): 657–62. PubMed ...Deleter Mouse in C57Bl/6 Based on FLPo Recombinase.” Genesis 48(8): 512–20. PubMed PMID: 20506501. 6. Logie,...

... enhanced GFP (EGFP), a variant with improved brightness (Tsien, 1998). Another mutation (A206K) is necessary...solution to avoid autofluorescence is offered by bioluminescent proteins. These probes generate light by a ...there is no need to excite the sample. Although bioluminescent probes are usually dim relative to fluorescent...recent engineering efforts have increased the brightness of these genetically encoded light emitters (...palette of Nano-lanterns for real-time multicolor luminescence imaging." Proceedings of the National Academy...PMC4394297. 16. Iwano, Satoshi, et al. "Single-cell bioluminescence imaging of deep tissue in freely moving animals...the Fluorescent Protein Collection Find empty backbones for fluorescent protein fusions Browse the Fluorescent...

...were able to introduce the desired mutations in genes associated with dementia, such as APP and PSEN1,...when screening reasonable amounts of single cell clones. Furthermore, CRISPR/Cas9 editing is often bi-allelic...2016). For those of you involved in the tedious business of iPSC clone picking, a 100-fold decrease is ...the difference between picking 100 versus 10,000 clones! We would therefore strongly encourage any researcher...intended sequence change and screen a few hundred clones to find the right one Step 2 – Edit the cells a...steps, you’ll be screening a total of a few hundred clones - Still better than 10.000, right? (Kwart, Paquet...incorporate mutations without screening thousands of clones to find a positive one. So if you have better things...

...their test genes, the researchers at the BIOFAB showed that expression of many different genes was predictable...coli. 800 and 1900 Series If you’re expressing two genes from a single transcript, or if you want to prevent...create the pCell-Free vectors, Gateway-compatible backbones that enable cell-free expression of proteins in... constructed a library of eGFP-tagged human ORF clones to allow testing and comparison of different cell-free...Cell-free expression test kit represents a set of 88 clones in a pCellFree vector that enables protein expression...enzyme function can lead to surprisingly large fitness effects during adaptive evolution of antibiotic...

...lifecycle. Halloween genes The Halloween genes are a set of cytochrome P450 genes discovered in Drosophila...Mutations in these genes are lethal, associated with defective exoskeleton formations. These genes were appropriately...1983): 813-816. Gilbert, Lawrence I. "Halloween genes encode P450 enzymes that mediate steroid hormone...

...takes several weeks to delete or deplete individual genes. Michael Laub’s lab developed a CRISPRi system to...downregulate expression of specific Caulobacter genes using a catalytically dead Cas9 (dCas9) derived ...Streptococcus thermophilus (the popular Streptococcus pyogenes system does not work well in Caulobacter). To ...system can also be used to downregulate multiple genes at once. Read the paper in mBio Find the CRISPRi...transgene design and delivery. They generated hPSC lines with AAVS1-integrated, inducible, and fluorescent...which increases activation of methylation-silenced genes. Read the paper in Nature Communications Find Casilio-ME...

...challenge when researchers are communicating across disciplines or doing different tasks. But these challenges...can be exaggerated when communicating across time zones, digitally, and asynchronously. The best way to ...collaborating with remotely. Depending on the willingness of the team to adopt various tools, this may ...collaboration includes scientists working in different time zones, it’s important that everyone can access data and... collaboration. Some things to figure out are timelines for experiments and manuscripts, who is responsible...your recurring meetings and reaching out if any milestones are missed. Spend time in person Presenting...

... so genes. It would be great if we could just go in and quickly manipulate individual mouse genes and ...genome editing system derived from Streptococcus pyogenes. An alternative Cas9, from Streptococcus aureus...effective at editing mouse embryos as Cas9 from S. pyogenes and has the advantage of being smaller (Zhang ...C57BL/6, for which we have germline competent ESC lines. Second, the process is much quicker. It takes 3...Wang is a professor at the Institute of Zoology, Chinese Academy of Sciences, and he is interested in using...Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated Genome Engineering. Cell ...

...thanks to our guest writer, Pamela J. Hines, PhD! Pamela J. Hines is a Senior Editor at Science magazine... post was contributed by guest writer Pamela J. Hines PhD, Senior Editor at Science Magazine. Although...replication and gene transcription during vertebrate embryogenesis. My interests, though, have proven unruly. Instead...journalism? Take a look at our news editors. A fondness for visualization of science? Look at our graphics...French horn, and rhubarb. Follow her on Twitter @Pam_Hines and LinkedIn. Additional Resources on the Addgene...

...graduate student, you are in a system that, quite honestly, does not value your potential contribution to... science, you are likely lacking the necessary business background and/or connections to even begin this...pitch what you hope to build.  Then ask for their honest opinions -- this part might hurt, but if you pitch...made, we considered the feedback and adjusted our business model accordingly until we had a model that was...This will allow you to discuss each project and business decision before making it, to have an internal...faculty, venture capital and angel investors, and business development heads at major companies.  The majority...

...more commonly used backbones and specific primers you can use to confirm your clones. You can also find...screen will help you more easily identify successful clones so you have to weed through fewer colonies after...antibiotic resistance. But what about identifying clones that contain your desired insert? This is where...how you can use restriction digest to screen your clones, but there are many other ways to choose restriction...PCR.   Sanger sequencing Sanger sequencing determines the precise order of nucleotides within a DNA...

...MSCV (Murine Stem Cell Virus) sequences. Packaging genes (viral Gag-Pol): Gag is a structural precursor protein...tropisms.  Packaging using helper-free packaging cell lines This method utilizes a packaging cell line that ...and/or Env, eliminating the need to deliver these genes in trans by specific plasmids. This method, therefore...standard γ-retroviruses use the Gag, Pol, and Env genes for packaging; however, they are different viruses...replication incompetent? Yes, as with lentiviruses, the genes required for γ-retroviral packaging and transduction...element; sequence that stimulates the expression of transgenes via increased nuclear export. LTR Subcomponents...

...nucleic acid-based vaccines. These vectors provide several advantages over traditional vaccines, including 1... knock it out, etc. It’s how we understand what genes do! Viruses have several options to allow you to...engineered to introduce frameshift mutations in genes of interest, turning expression off permanently....2007.   Clinical applications Viruses in vaccines helped pave the way for their use in other clinical...The use of viral vectors in vaccine development. Vaccines. 7, 75 (2022). DOI: https://doi.org/10.1038/s41541...

...virus will target and fuse with. In nature, this determines which organisms and cells they infect. In the...the fear of random genomic integration causing mutagenesis in the host. Adenoviruses can deliver relatively...Dividing Cells Moderate Immunogenicity Insertional Mutagenesis Possible Lentivirus Infects non-dividing...non-dividing cells Insertional Mutagenesis Possible Adenovirus Remains Episomal (Little Risk of...of Insertional Mutagenesis) High Gene Expression Infects Non-Dividing Cells Large Packaging Capacity (... AAV Remains Episomal (Little Risk of Mutagenesis) Very Low Immunogenicity Infects Non-Dividing...

...all known genes. This is a perfect example of an unbiased, reverse screen – all known genes are assayed... a biological process. Players can be proteins, genes, enzymes, drugs, or anything else that plays a role...where there is removal of known factors (such as a genes) and the loss of those factors is assessed. The ...similar to CRISPR, but instead of knocking out the genes of interest (or the whole genome), they knock down...especially useful if you are interested in studying genes whose knockout would be lethal to cells and thus...

...on cell lines susceptible to or dependent on certain media components, and are rescued by genes supplied...colonies (used to complement auxotrophic cell lines), or genes which rescue a lethal phenotype. Resources... transformed plasmid. Such plasmids may contain genes that allow for the use of a particular substrate...list of Bacterial Expression Systems Find Empty Backbones for Your Experiments    ...

...number of genes involved in cardiovascular and metabolic diseases. We are knocking out genes that have...mutant versions of 15 different disease-associated genes. Addgene spoke to Musunuru about how the new kit...kit is quite useful in generating the mutant cell lines that one needs to do disease modeling in stem cells...several mutations in the same gene or in multiple genes, all of which seem to contribute to a particular...iPS) cells from patients and try to compare those lines, they may all have the mutations but they will also...