...blogger Matteo Tardelli! Matteo Tardelli, PhD is Postdoctoral Scientist at Weill Cornell Medicine in NYC...was contributed by Matteo Tardelli, a postdoctoral scientist at Weill Cornell Medicine. Discussing data...Hamilton DP, Denfeld BA, de Eyto E, Hampton SE, Keller PS, Sharma S, Lewis ASL, Weyhenmeyer GA, O’Reilly...

...now.  Many thanks to our guest blogger, Kelly Hills! Kelly Hills is a founding bioethicist of the consulting...This post was contributed by Kelly Hills, founding bioethicist of Rogue Bioethics. On November 25, the... the MIT Technology Review dropped a bombshell report. A scientist working in China was using CRISPR/Cas9...separate things happened nearly simultaneously: The Wellcome Trust funded a center for mitochondrial research...consulting firm Rogue Bioethics. Kelly utilizes her firsthand experience in the software industry to inform...

...multispecies biofilm formed by Pseudomonas spp. and Klebsiella spp. Dr. Devarshi Gajjar, The M. S. University...research focuses on a collection of pathogenic Klebsiella and Pseudomonas isolates causing urinary tract...unknown regulatory regions in Pseudomonas and Klebsiella spp. The effects of these deletions on biofilm...major pathophysiological complications of diabetes mellitus. Recombinant human proteins that play a role in... Researcher who investigates the molecular and cellular etiopathomechanisms of ischemic stroke, the most... reduced blood flow. Novel treatment options, as well as non-pharmacological solutions to modify the course...

...of viruses used to deliver CRISPR components to cells or organisms under study. In the past we’ve left... they want to do is to quickly generate a single cell line and proceed to use it for experiments.” - ...screen for genes involved in a wide variety of cellular processes. While these are widely requested and...representation in the viral prep, infect target cells, and, finally, conduct the screen. To help alleviate...of this labor for our users, we now provide the Brunello (targeting human genes) and Brie (targeting mouse...deliver gRNA and therefore, must be used to infect a cell line already expressing Cas9. As John Doench (Broad...

...negative bacteria is due to differences in cell wall composition. Cell wall structure Gram positive bacteria...used in the staining process bleached another, Klebsiella pneumoniae. After realizing this was a repeatable...membrane. Common gram negative bacteria include salmonella and E. coli. Because of their thicker peptidoglycan...complex within the peptidoglycan layer, staining the cell purple. When the alcohol is added, it dehydrates...Silhavy, TJ., Kahne, D. & Walker S. The Bacterial Cell Envelope. Cold Spring Harb Perspect Biol. 2(5) a000414...

... between Cas9 (which causes the breaks) and host cell DNA repair factors (which repair those breaks) makes...example, correcting the allele that causes sickle cell anemia. However, HDR occurs much less frequently...a structure could drive sequence replacement in cells. Targeting three dCas9 molecules to a precisely ...functional Cas9 protein and the targeting guide RNA into cells by electroporation. Results from our lab and others... HDR. The detailed protocol for nucleofection as well as the sequences of our validated editing reagents...Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins...

...government agencies, active researchers, as well as fellow iGEM teams. We were the proud hosts for the...industrial or research purposes requires stable host cells (the simplest example would be a transformed mesophilic...successful) as host systems since their biology is well studied, and they are convenient to maintain under..., since mesophilic chaperones would not function well under suboptimal conditions. This means recombinant...CRISPR-Cas mediated editing for psychrophilic systems as well as engineering and regulating psychrophilic recombinant...

...address all your unanswered questions via text! (well, maybe not all of them. We still haven’t gotten ...and would like to get it working in microglia as well. A:  We are not aware of any available AAV for efficiently...co-delivering different AAV particles into the same cells in vivo? A: This will be highly dependent on vector...particularly one that can target the epithelial cells in the blood-brain barrier?   A: For liver, we recommend...worth investigating for both Q6 & Q7.For epithelial cells of the blood brain barrier we would currently recommend...and to all our participants for their time and excellent questions.    Additional Resources on the Addgene...

...stimulate AraC binding to I1 and I2 sites. Supplementing cell growth media with glucose decreases cAMP and represses...Steroid-regulated LexA promoter XVE (synthetic) pFZ19 (yellow flame) Temperature inducible promoters Temperature...gene transcription to take place. Addgene also has yellow flame light-regulated two component systems designed... Because transcription machinery differs between cell types or organisms, promoters are similarly variable...variable. Bacterial promoters only work in prokaryotic cells and typically only in the same or closely related... were derived. Similarly, the various eukaryotic cell types (mammalian, yeast, plants, etc.) require unique...

... into the cell. This means that not all neurons and pathways will be transduced equally well by AAV-retrograde...they either trace downstream from the soma (neuron cell body) toward the axon (anterograde tracer) or upstream...them useful tracers, but finding a virus that works well for retrograde tracing can be difficult. The in ... Data Hub!) The cortical pathways to the IC were well transduced by AAVrg while other pathways such as...chemogenetic vector directly into the region where the cell bodies of the projection neurons reside.   Impact...

...electrocompetent cells in which the phage recombination machinery is induced using L-arabinose. These cells can be...function of certain regions in a promoter or gene, as well as to study the effects of inactivating/activating...coli to undergo nick repair. This method can work well, but poor template digestion can result in a high...required in recombineering. REPLACR-mutagenesis is also well suited for insertions and deletions. The figure ...Enzymes Required No No Yes Yes Lab Made Competent Cells Required Yes No No No Comparison of REPLACR-mutagenesis...

...also gives cells antibiotic resistance to neomycin so that you can select the population of cells with the...transcription factor. The FLAG antibody has been well characterized and the system allows you to add this...Only a small percentage of the proteins inside a cell have a commercial antibody against them. There’s...antibody. It’s an epitope not found in human or other cells. It was made artificially. That means you aren’t...lab already should be able to run this protocol as well. We would also like to put some social pressure ...

... be expressed in the same cell, but not necessarily at the same subcellular compartment as the opsin or...modulate the activity of neurons and other brain cells. Since the opsins or chemogenetic receptors used...neuropil conceals identification of opsin-expressing cells). For other things to keep in mind when making fluorescent...cerevisiae by use of an epitope addition method. Mol Cell Biol 8:2159–2165 . https://doi.org/10.1128/mcb.8.5.2159...Paré J, Bonaventura J, Richie CT, Michaelides M, Mueller SAL, Roseboom PH, Oler JA, Kalin NH, Hall RA, Smith...

...we virtually always use every single well in a 96-well (or 384-well) plate! How much do repositories help...in this post is a mathematical story well beyond my ability to tell. But even from these rough estimates...depositors have helped us save the scientific community well over $80 million on plasmids alone. (And we expect...

...product, the transformation may not result in any cells, or all the colonies screened might not contain ...i.e. primer3) or provided by the major companies selling PCR reagents. Nowadays commercial polymerases are...verify the strain and the efficacy of your competent cells (when they get old, they lose competency). If all... look at your experimental design. Full lawn of cells. The antibiotic in your plate might not work. Check... the incubator. The small colonies are called satellite colonies and they form when all the antibiotic...acknowledged in hundreds of publications including Cell, Nature, and other journals.   Paolo Colombi is ...

...-HF1, & HypaCas9. The off-target problem It is well known that CRISPR/Cas9 genome editing can result... the EMX1 locus in human embryonic kidney (HEK) cells, 11 of these initial mutants retained the on-target...off-target activity at multiple genomic locations in HEK cells. Further testing of two of the mutants, SpCas9(K855A... showed that these mutants are not toxic to HEK cells and that similar mutations can improve the specificity... researchers to make precise edits in mammalian cells and may decrease worries about off target effects... Cas9 in complex with guide RNA and target DNA." Cell 156.5 (2014): 935-949. PubMed PMID: 24529477. PubMed...

...process will begin. Partnering with seqWell, we are using the plexWell technology to easily and quickly ...ever worked to sequence DNA on NGS platforms will tell you, while the process has become much more refined...assembly process. Again, thanks to our partners at seqWell we make use of a pipeline that takes the raw data...necessary adapter sequences for Illumina sequencing as well as unique barcodes per sample so we can assign sequencing...Tasissa M, Kostman M, Tillgren M, Makana Hanley L, Mueller I, Mitsopoulos A, Fan M (2020) A Novel Next-Generation...

...Communications, Harvard University Ray Howell -Founder of Howell Communications Philip Brenner, PhD - Assistant...scientific enterprise and the way it is funded have been well documented over the past 2 years (Alberts et al,... to shape the sector throughout their careers (McDowell et al, F1000Research, 2014; Dolan, F100Research...relationship, along with, of course, union dues. Time will tell the extent of the implications of this ruling and...27175853. Pubmed Central PMCID: PMC4866822. 3. McDowell, Gary S., et al. "Shaping the Future of Research...

... acid (DAP), a component of the cell wall. Once in the recipient cell, the CRISPRi DNA (purple) flanked... is propagated during normal DNA replication and cell division. Mobile-CRISPRi does not disrupt gene function...mobilize these plasmids for transfer to recipient cells during a tri-parental mating scheme. Mobile-CRISPRi...bacteria associated with human disease (S. aureus, Klebsiella pneumoniae, and P. aeruginosa) in addition to...antibiotic trimethoprim. When Enterobacter aerogenes, Klebsiella pneumoniae, and P. aeruginosa (all opportunistic...

...AmpliCan We chose to adapt Crispresso by Luca Pinello and Kendell Clement for batch analysis because of its...Leonetti Ryan Leenay Jason Li Max Haeussler Luca Pinello Kendell Clement Andy May Additional resources on the...members have taken up to a month to design a single 96-well plate for HDR that satisfy all requirements. To ...all the standard information that goes into a 96-well plate and comes out of it. Biotech companies are...