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... The previous post in this series described a practical approach to selecting a bright fluorescent protein... localized fusion proteins and do not require dedicated plasmids or constructs. It should be noted that...reached at high power may not translate to real applications in which far less power is used. For example...insight into the photostability in the intended application (Goedhart et al, 2012). The third issue relates...Selecting a photostable protein The issues with quantification of photobleaching rates can be summarized as...Amsterdam). He develops, characterizes and uses genetically encoded fluorescent probes. You can follow him...(University of Amsterdam) where she develops genetically encoded FRET based biosensors.     Additional...

...by guest blogger Alan Wong. The complexity of biological systems can hinder our attempts to study and ...combinatorial genetic networks underlying your favorite biological phenotype in a scalable way. This blog post will... genetics that underlie complex regulation in biological systems. The CombiGEM platform provides a means...sequences via oligo synthesis using the format indicated below and pool-clone them into the pAWp28 storage...realization of a variety of perturbations and applications in functional genomics, cell reprogramming, ...currently an Assistant Professor at the School of Biomedical Sciences of the University of Hong Kong. He is...technologies to interrogate and understand complex biological systems. He can be contacted at [email protected]. ...

...As evidenced by all the CRISPR publications, press, and plasmids out there, it’s obvious that CRISPR ... Not only is CRISPR having effects on various biological disciplines, the base technology itself is constantly... the Broad Institute, the Joung lab at Harvard Medical School, and the Doudna Lab at Berkeley set out ...afforded by gRNA binding at non-target sites would theoretically decrease off-target cutting. Figure 1...PubMed PMID: 26628643. Find plasmids from this publication at Addgene. 2. Nishimasu, Hiroshi, et al. "Crystal...PubMed PMID: 26735016. Find plasmids from this publication at Addgene. 5. Chen, JS, et al. "Enhanced proofreading...PubMed PMID: 28931002. Find plasmids from this publication at Addgene. Resources on the Addgene Blog Read...

...spacer "NNNTANNN", where "N" indicates which bases may vary from the canonical sequence. Among these are ...important plasmid elements – promoters, origins of replication, protein tags, and antibiotic resistance markers...creating (excuse the pun) specific, targeted DNA modifications in transgenic animals, embryonic stem cells,...proteins or co-factors, thus allowing for broad applications in a variety of experiments. LoxP (locus of ...sequence gives the loxP site directionality, and the canonical loxP sequence is ATAACTTCGTATA-GCATACAT-TATACGAAGTTAT... the loxP methods described above, a genetic modification can be restricted based on experimental constraints...

...are time-consuming, have been typically optimised for immortal/genetically-corrupt cell lines, lack scalability...alone in the cellular milieu. It is, therefore, critical to understand how proteins interact with one another... another (or with DNA) to perform diverse biochemical tasks in the cell. One of the most popular approaches... to tag the protein of interest with a small biochemical “epitope tag” such that these tagged proteins... purchased from various other suppliers with a typical turnaround time of 5-7 days. The crRNA and tracrRNA... to tag >30 genes/plate in the first shot with typical knock-in efficiencies of 10-20%. Together, our ... the non-HDR allele. For N-terminal tagging, specifically searching for error-free knock-in at both alleles...

...ago. I just recently started working in the pharmaceuticals industry after earning my Master’s in 2016 ...accomplishments and demonstrate your science communication skills. Know your audience. Connect your prior...presentation as an opportunity to demonstrate technical and transferable skills that make you qualified...perfect science talk Be prepared for potential technical difficulties. Have your presentation on a flash...codes for interviews. You can also ask HR for clarification on the dress code for the interview. Greet each... quiet place to take your video call.  Expect technical difficulties. To try and circumvent this, test...from HR carefully. And even if you don’t have technical challenges, your interviewers might.  You’ll probably...

...named according to the number of strands in the canonical beta-barrel structure. Most frequently, the proteins...pairs with N173 derived from Cerulean or Venus Identification of new fluorescent protein fragments for bimolecular...fluorescence complementation analysis under physiological conditions. Shyu et al. Biotechniques. 2006 ...fluorescent proteins for protein labeling and signal amplification. Zhou et al. PLoS One. 2020 Bo Huang Yellow...Yellow Venus pBiFC-VN173, pBiFC-VC155 Identification of new fluorescent protein fragments for bimolecular...split-FP options. While the BiFC interaction is typically irreversible and requires some time for chromophore...

...methyltransferase DNMT3A allows researchers to easily add biochemical marks to genetic regions from which where they...approach (spoiler alert: it’s dCas9… with no modifications). Ready to get started?   Figure 1:...eventually, develop treatments, we need tools to specifically edit methylation sites.  Step aside, transcriptionally... DNA. When dCas9 is bound to a target CpG, it physically blocks DNMT1 from that site. So, when DNMT1 methylates... methylates the genome during DNA replication, it doesn’t methylate sites blocked by dCas9. Within a few...Szyf 2021.   Let me leave you with a few practical considerations: Design your gRNA to overlap the...for dCas9 to interfere with DNMT1 even through replication. The dCas9 also needs to be removeable, otherwise...

...process involves a significant amount of QC. First, we perform Picogreen quantification on each and every... preparation process and includes Picogreen quantification of various intermediate steps, a sizing electrophoretic...electrophoretic gel, and qPCR quantification of the final libraries. A broader view of the whole plasmid...often find a few mismatches in the origin of replication or other common backbone elements. Since we’ve...annotated Genbank files that are useful for more complicated plasmids, like those that contain synthesized...sequence or plasmids containing genes with many modifications. We look for point mutations, truncations, and...

... from E.coli, named TadA7.10 in ABE7.10)  a catalytically impaired Cas protein (dCas or Cas nickase)  ...accidental insertions and deletions (indel) by the catalytically impaired D10A nickase from ABE7.10 can be a ...the authors created ABE8 constructs using a catalytically “dead” S. pyogenes Cas9 (dC9-ABE8.x-m/d). The...even in adult patients. In order to analyze the clinical use of ABE8 for primary human cell editing, the... reduced off-target editing frequencies. For applications that require high DNA editing specificity, for...deamination, an important attribute for therapeutic application. ABE8s demonstrate an overall improved base editing...editors with increased activity and therapeutic application. Nature Biotechnology. https://doi.org/10.1038...

... Roska’s lab at the Institute of Molecular and Clinical Ophthalmology Basel recently took a different ...Strategy #2 (ProB): Create promoters using phylogenetically conserved sequences that are upstream of the...expression. Surprisingly, <1% of synthetic promoters replicated the expression specificity of the wild-type promoters...reproducibly labeled retinal cells can be found here.  Applications for synthetic promoter AAVs Besides restricting...synthetic promoters have a few other potential applications: Using promoter combinations for targeted expression... promoters expressed in. The lab tested this application using one promoter to drive expression of Cre-mCherry... rates of labeling are ideal of gene therapy applications, promoters that produce sparse labeling would...

...progress on establishing a system for the chemical replication of RNA in the absence of evolved enzymatic...enzymatic machinery (i.e. RNA replication with no proteins). The chemistry is complex and this work requires...inheritance that might be less error prone than DNA replication. He too mentioned how “surprising” small RNAs... about using CRISPR/cas to address the growing clinical problem of antibiotic resistant bacteria. See ... and Sun Yat-sen University presented two new technical developments. Using the Tet-on system, his lab...Steitz quipped that RNA in the ribosome is in a complicated conformation “not like DNA which has a boring...

...Because of their small size, nanobodies can be synthetically expressed off of plasmids in bacteria, and they...with a large and diverse pool of sybodies that hypothetically contain ones that bind strongly to your target...Starting with libraries and a biotinylated (for purification purposes) protein target of interest, the protocol... identifying sybodies that bind strongly and specifically to the protein of interest. The Seeger lab used...-30) of sybody candidates are sequenced for identification. All unique sybodies can be subcloned into ...2020) Generation of synthetic nanobodies against delicate proteins. Nature Protocols 15:1707–1741. https...Light to Manipulate Cell Signaling and Protein Purification RANbodies: Reporter Nanobody Fusions Plasmids...

...McDonald. "A practical guide to evaluating colocalization in biological microscopy." American Journal of... proteins of interest and examine them using biochemical methods thus removing the context of the cell...way it is important to choose the appropriate quantification methods and tools. It is usually not enough...microscopy is limited by the wavelength of light and, practically speaking, for common lab microscopes the detection...Quantitative analysis of dynamic association in live biological fluorescent samples." PloS one 9.4 (2014): e94245...

... (green or blue) within a population of timer indicates the age of the pool: the higher the ratio, the...fluorescence falls within the linear window for the theoretical timer shown in Figure 1B, you can use the equation...-FT-N1 pTRE-Slow-FT pBAD/HisB-Slow-FT -  Applications of fluorescent protein timers FP timers have...protein, or cellular structure and a particular biological event (trafficking to a subcellular location,...of neural fiber bundle formation." Journal of Biological Chemistry 276.32 (2001): 29621-29624. PubMed ...to red report on cellular trafficking." Nature chemical biology 5.2 (2009): 118-126. PubMed PMID: 19136976... insulin-secreting beta-like cells." Nature communications 7 (2016). PubMed PMID: 27063289. PubMed Central...

...requires incubation at 55 °C - a dangerous, non-physiological temperature for mammalian cells and one which...been excitedly testing NgAgo's genome editing applications. So, how does NgAgo stack up thus far? A few...survey. When asked if they could detect indels (an indication of gene disruption ability) with NgAgo, one respondent...could not achieve indels with the NgAgo system indicates that it is very tricky to use and fussy to optimize...proposed in the original article, to achieve greater efficacy, researchers will have to carry out repeated transfections...results out there include: Google Groups - I've specifically set up a forum where NgAgo users can share protocols... S. Lander, and Feng Zhang. "Development and applications of CRISPR-Cas9 for genome engineering." Cell...

... most commonly used are GECIs - genetically encoded calcium indicators (e.g. GCaMP). Others include: pHluorin...capacity. Many of these FPs are fluorescent when ectopically expressed in most organisms. Furthermore, fusing...fluorescence. Therefore, FPs are used to study many biological questions. The two most common uses are: 1) to...For LSS proteins, the Stokes shift is ≥ 100nm. Specifically, these proteins are excited by UV light or blue...were prone to oligomerize. This may affect the biological function of the FP-fusion protein. Therefore,.... al. (2010) “Fluorescent proteins and their applications in imaging living cells and tissues”  Physiol...

...excited to announce we now offer an export license application for rabies materials! Some of the materials in...international requestors. We also offer export license applications for VSV and SARS-COV-1 plasmids. Export control...will need to provide a brief description of the applicable research project when purchasing export-controlled... applied to cover processing costs. Once the application is approved, Addgene then ships the material ...offered VSV and SARS-COV-1 plasmids under this application process since May 2020. We find this process ...whenever possible, as we cannot expedite the application process.  More information about export control...

... antibiotic resistance genes so that you can specifically isolate bacteria containing your constructs ...common antibiotic resistance genes and which applications are best (or worst) for each antibiotic. The...Ampicillin Ampicillin, often just called amp, is the historical king of the antibiotic resistance realm. Amp ...immediately after it has been heat-shocked or electrically zapped. As a result, a shorter recovery period...is expensive (relative to amp), and for some applications, the benefit it offers is marginal. Fortunately...antibiotic resistance ultimately comes down to your applications and needs. The more niche and specific your ... antimicrobial agents in MIC trays. Journal of Clinical Microbiology, 23(5), 959–961. https://doi.org/...

...translational applications (mechanistic studies and gene therapy treatments for neurological diseases). ...neuroscience research is the lack of a method for genetically manipulating neurons throughout the whole brain... creates a template that’s suitable for PCR amplification using pre-designed primers. By using this library...astrocytes. Why screen for IV deliverable AAVs? Typically AAVs are directly injected into the brain, but...throughout the body and is often difficult to surgically access. Therefore an IV injection is an ideal...pictures (see figure 4). It also enables single-cell anatomical study and neuronal tracing, aka determining where...