...right CRISPR vectors on Addgene and found instead ones that were… pretty close, or at least close enough...Fragmid depositor John Doench reports even the greenest members of his lab, high school interns, were ...created over five months. When they tested those clones (in duplicate), 93% had the correct banding pattern...enzyme digest. They sent 82 of the correctly banded clones for plasmid sequencing and found 98% of the plasmids...Drosophilia Plasmid pRDB_051 (for stable fly cell lines; attL and attR sequences for genome integration ...Drosophilia Plasmid pRDB_052 (for stable fly cell lines; Hygromycin resistance gene; for genome integration...own Fragmid components! Ensure your backbone and genes contain only the Bbsl cut sites needed for the Fragmid...

...regulate the transcription of specific genes. What if one of those genes coded for a protein that regulated...combinations of different candidate genes at once to identify the best cocktail of genes when the relationship between...Level 0: Cloning of basic parts (promoters, UTRs, genes, tags, etc.) into MoClo-compatible vectors. Level...the DNA that encodes the protein itself or other genes within the circuit. These interactions yield a cellular...stacking is the use of a combination of multiple genes to achieve a desired effect. This is commonly used...

...ex: rods or cones) or cell classes (ex: photoreceptors which encompassed both rods and cones).  Targeted...of cell types, such as inhibitory neurons, rods, cones, or retinal ganglion. Rather than using naturally...of the start codon of retinal cell-type specific genes from mice. Strategy #2 (ProB): Create promoters ... transcription initiation sites of at least two genes with high expression and high cell-type specificity...to loosely match the specificity of their source genes, i.e. if a synthetic promoter expressed in cells... Transgenic animals allow for the expression of genes in particular tissues, but they are costly and time-consuming...

...also vary in their brightness, depending on the respective fluorochrome. Brightness is impacted by both...the background. Dyes that can pass through cell membranes can prove useful for live-cell imaging. Experimental...maintained through mechanisms like fission, fusion, biogenesis, and mitophagy, can be tracked using dyes like...for super-resolution usages, such as superior brightness, photostability, and most importantly, a photoswitching...combination is Alexa594, and Alexa647 due to their brightness and high degree of overlap. Dyes for DNA/RNA ...The field of fluorescent ligands and dyes has witnessed remarkable advancements, driven by the need for...receptors. In Biochimica et Biophysica Acta - Biomembranes (Vol. 1838, Issue 1 PARTA, pp. 15–33). https...

...sequencing of puromycin resistant clones, they found that 80% and 50% of clones displayed proper insertion at... Addgene depositor Takashi Yamamoto’s lab has harnessed MMEJ to create a new method for CRISPR gene knock-in.... Using their PITCh plasmids, GFP knock-in cell lines can be created in about a month and a half, without...promoter, and the technique can be adapted to other transgenes. It’s important to note that the fluorescence... would be advantageous to adapt PITCh to insert genes into AAVS1, the “safe harbor locus” of the human...and Ling Lu. (2016). “Highly efficient CRISPR mutagenesis by microhomology-mediated end joining in Aspergillus...

...organisms that can't reproduce with wild ones. For example, Drosophila lines with compound autosomes are completely...populations. Whereas most genomic changes impose a fitness cost and are eliminated by natural selection, gene...didn't work quite as well as anticipated4, greater awareness will certainly improve safety. What safeguards...? There are ongoing efforts to develop formal guidelines to answer this question. Until then, using multiple...guest blogger! An inventor of technologies that harness evolution, Kevin Esvelt studies ways of using molecular...www.sculptingevolution.org. References: Burt, A. Site-specific selfish genes as tools for the control and genetic engineering...

...has always been focused on gene transfer, from transgenes to plants, chloroplast to the nucleus, or pathogens...The MoClo Plant Kit makes it really easy to clone genes for expression in plant cells. Sylvester and I chose...includes everyone’s favorite promoters, plus a few new ones that are not used so much but have comparable expression...and give similar stability to transcripts as the ones that people use all the time. Now there are many...to a lot of pathogens. It’s also easy to express genes in, which makes it a good laboratory workhorse. ...Tim-Martin Ehnert, Stefan Werner, Jonathan D. G. Jones, Nicola J. Patron, and Sylvestre Marillonnet. ACS...Plant SynBio Reading: Quick, Versatile Plant Transgenesis with GreenGate Plasmids  ...

...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 ...

...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,...

...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...

... 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...