...Forster Resonance Energy Transfer (FRET). Through the non-radiative transfer for energy between neighboring...Whether BiFC constructs are expressed transiently by transfection or stably impacts the success of BiFC...a dose-dependent manner. This is straightforward in easy-to-transfect cell lines where plasmid dosage ..., FRET can be used to detect the inter- and intramolecular interactions that underlie protein function..., kinase signaling, integrin signaling, and transcription factor interactions (Kerppola, 2006). One of...interactions between basic leucine zipper and Rel transcription factors (Hu et al., 2002). This would be more...subcellular localization of the studied PIPs. The straightforward nature of BiFC also makes it easier to generate...

...frankenbody can be effectively used to track single mRNA translation in U2OS cells. HA is small, only 9 amino...Stability improvement of antibodies for extracellular and intracellular applications: CDR grafting to stable...based probes the Stasevich lab used an alternative strategy to develop a genetically encoded antibody based...which contains a 10xHA tag. These experiments demonstrated that the HA frankenbody can be used to amplify...minutes in living cells, meaning that scientists can track the movement and localization of single HA-tagged... amino acids, thus it is translated quickly by the ribosome. This feature provides HA frankenbody the ...researchers used the HA frankenbody to successfully track nuclei in a developing zebrafish embryo over an ...

...ready-to-use viral preps   Improved cell-free transcription/translation system to examine biosynthetic pathways... Gabrielle Clouse Recently, cell-free transcription/translation systems (also referred to as TX-TL systems..., the DNA of interest, and cell extract.    Graphical Abstract: Schematic representation of cell-free...the expression of fluorescent proteins and oxytetracycline enzymes. Next, this system was used to recreate...modifications to edit DNA without requiring a double-stranded break. The Xiao Wang Lab has recently deposited.... These plasmids use “Cas9-mediated adenosine transient reporter for editing enrichment”, or XMAS-TREE...modifications and work with them further. The lab demonstrated that XMAS-TREE can be multiplexed and can be...

...C2c3 are related to Cpf1, but they require both a tracrRNA and crRNA to cleave target DNA (Cpf1 requires ... Cas13a binds and cleaves RNA rather than DNA substrates. In terms of structure, Cas13a shares no homology...PAM (PFS) Cleavage Mechanism Cas9 HNH, RuvC TracrRNA, crRNA DNA 5' NGG Specific blunt ended DSB...endogenous CRISPR/Cas9 system, Cas9 uses both a tracrRNA and crRNA to facilitate binding and cleavage of...the crRNA or presence of a PFS. This is in stark contrast to Cas9, which requires that each DNA target have...Recombinase Polymerase Amplification (RPA) or Reverse-transcription RPA (RT-RPA), respectively. Amplified nucleotides...have utilized dCas13 fusion proteins for imaging, tracking, modulating splicing, and regulating expression...

...cytosol (instead of on the yeast extracellular surface) which enables tracking of protease activity with a ...protease allows a transcription factor to enter the nucleus and modulate transcription of the mCitrine ...affinity variants of TEV. The new system has been demonstrated to work for evolution of other target proteins...structures within cells. HeLa cells were transfected with plasmids encoding FPs tagged with the plasma...biotin-4-fluorescein to be dissolved at a higher concentration, and results in uniform NB biotinylation. Once...expression can vary significantly depending on transgene design and delivery. To overcome these variations...reporter. With this system, they could quantify and track cells expressing both dCas9 and gRNAs. The...

...Healthcare consulting firms that specialize in business strategy tend to be mostly engaged by pharma and biotech...Consultants can help these teams develop communication strategies, or understand, and publish on the health and...need help with the intricate task of designing a strategic vision for a portfolio of candidate drugs. A decision...from previous consulting experiences, educational training, and conferences, and synthesizing this knowledge...have greatly benefited from and appreciated the training I’ve received in concisely and accurately communicating...and work styles (e.g., Myers-Briggs). Staff are trained on how to work best with diverse teams, to leverage...of different personality types, and to devise strategies to be prepared for addressing unexpected situations...

...expression cassettes Traditionally, generating expression cassettes for yeast transformation can take 5 days...covalently joined, scarless DNA – perfect for transforma­­­tion or as a PCR template. Some of the applications...by using NEBuilder which requires only a single-stranded synthetic oligo to generate a single-guide RNA...RNA (sgRNA). Using only one single-stranded oligo, instead of the more typical two oligos, is more cost...annealing step usually used to create a double-stranded oligo. Generating an sgRNA construct becomes as...target region. Assembled plasmids can be directly transformed into E.coli for plating, screening and generation...inserted into a linearized cassette, ready for transformation into yeast that same day.  The field of synthetic...

...repeat region. B) RNase III, the tracrRNA and Cas9, bind to these transcripts and C) cleave them leaving mature...leaving a F) double-strand break. A “gRNA” is a researcher-designed hybrid of the tracrRNA and the crRNA. ...loci. 3. Cleave target DNA resulting in a double-strand break (DSB) Cas9 and its variants have two endonuclease...positions the nuclease domains to cleave opposite strands of the target DNA. Thus, the end result of Cas9...which defines the genomic target for Cas9, and the tracrRNA which acts as a scaffold linking the crRNA to ...for processing pre-crRNA into mature crRNA and tracrRNA binding. The spacer regions, on the other hand..., the “direct repeat region” combined with the tracrRNA forms the scaffold portion of a gRNA and the “...

...fluorescent proteins whose spectra overlap, there are techniques, like spectral unmixing, which can be used...Chroma Spectra Viewer comparing a standard Cy3 or Rhodamine filter set (Chroma #49004) to the spectra of ... the lighter colors; excitation spectra are blue and emission spectra are red. Neither is a perfect match...fluorescent proteins whose excitation and emission spectra differ sufficiently for them to be imaged in distinct...requires knowledge of their excitation and emission spectra, but a good rule of thumb is that both the peak...will want to compare the excitation and emission spectra of your protein with the filter sets or lasers ...emission filters and the excitation and emission spectra of the protein, so that the protein is well excited...

... when supplied with a luciferase substrate, the luciferase transfers energy to the FP, resulting in a ...luminescent substrate is required for imaging, and it is important to verify that this substrate does not...luciferase has a low quantum yield, and when transiently expressed in cells, generates much less signal... principle of bioluminescence resonance energy transfer (BRET). Photons emitted by a Renilla luciferase...excitation light source. Instead, a luciferase substrate like coelenterazine is supplied in the culture...labeled tumors in freely moving mice injected intravenously with coelenterazine, with increased sensitivity... which is limited by depletion of luminescent substrate, they synthesized diacetyl coelenterazine-h. Unlike...

...use of pre-existing transgenic lines, termed BLITZ (Biotin Labeling In Transgenic Zebrafish) (Xiong, et...“single-use” transgenic lines. Instead, researchers can generate and maintain transgenic lines that can...protein by a specific transcription response element (TRE) or kinase translocation reporters (KTRs). Find...interest and, for in vivo work, generate a dedicated transgenic line. Using zebrafish as a model organism, the...protein can then be co-expressed in essentially any transgenic zebrafish line expressing a GFP-tagged version...generational evolution of the AAV capsids (left) and transduction patterns (right).  Both AAV-PHP.eB and the AAV.CAP-B...mutations in mammalian cells. Long sequences traditionally have higher risks of off-target mutations. These...

...Murray’s lab uses cellular extracts to perform transcription and translation in vitro, and the new BioBuilder...handful of local high school teachers receiving training modules in synthetic biology has grown into a ...cutting-edge research projects in synthetic biology and transforms them into modules that students and teachers ...droppers, centrifuge tubes, circuit parts, and lab-strains of bacteria to study — to conduct their hands-on...and test hypotheses. Students then use cellular extracts as a means to rapidly prototype DNA circuits and...resulted in a new BioBuilder book chapter and an illustrated, downloadable classroom poster — with both of...scientist mentors and the BioBuilderClub community. Registration is open through October 19. The BioBuilder Learning...

...increase as the DNA amplicon transitions from single-stranded to double-stranded, and finally, drop once more... by abundant transcripts, leading to falsely lowered levels for less abundant transcripts. However, adding...can even be used to detect a single copy of a transcript. But this sensitivity can sometimes cause as ...preferably weighted towards the ends of the primers. Concentration of primers should be between 100 and 400 nm ...optimal annealing temperature and optimal primer concentration; check for primer dimers; and validate assay...signals are produced by the presence of double-stranded DNA, when temperatures are high, signal intensity...reflecting the presence of primarily denatured (single-stranded) DNA. As the temperature drops, single intensity...

... signaling pathways and the linkage of intra- and extracellular environments. Found on nearly every cell...eight beta subunits. Both subunits have short intracellular ends and large ectodomains containing ligand-binding...the cell membrane, physically connecting the extracellular environment and the cytoskeleton. Most integrins...affinity for ligands. When integrins interact with extracellular ligands, their ectodomains can extend and open...mediating cell adhesion and migration, initiating intracellular signal cascades, and allowing agile responses...The actin cytoskeleton exerts tensile force, transmitted through the integrin, where it is resisted by...by the ligand embedded in the extracellular environment. This further stabilizes the extended open integrin...

... avidin, proximity labelling can capture weak/transient protein interactions as well as be used for both...after BioID became available, the Alice Ting Lab demonstrated that engineered ascorbate peroxidase (APEX),... is an enzyme-mediated proximity cell labeling strategy to detect cell-cell interactions in vivo (Ge et...2019). This technique relies on an S. auerus transpeptidase sortase A variant (mgSrtA), which is capable...36.9 (2018): 880. PubMed PMID: 30125270. PubMed Central PMCID: PMC6126969. Che, Yonglu, and Paul A. Khavari...27(8), 1188-1196. PubMed PMID: 26912792. PubMed Central PMCID: PMC4831873. Lam, Stephanie S., et al. "Directed...12.1 (2015): 51. PubMed PMID: 25419960. PubMed Central PMCID: PMC4296904. Martell, Jeffrey D., et al. ...

... on size is ultracentrifugation. Sedimentation equilibrium analytical ultracentrifugation yields an affinity... In corals, the red fluorescent proteins form tetramers. In general, fluorescent proteins have a natural...in some applications (e.g. labeling of cells or tracking promotor activity), but it is problematic in applications...oligomers can be analyzed by several techniques. Gel filtration and SDS-PAGE under non-denaturing conditions ...will depend on the homodimer affinity and the concentration of the protein solution. During the experiment...affinity and has been used to demonstrate that yellow fluorescent protein homodimerization is characterized...Zacharias et al, 2002), meaning that at this concentration 50% of the protein forms dimers. Finally, spectroscopic...

...microenvironment. This results in increased expression of transforming growth factor-β signaling, possibly inducing...tumor microenvironment is the immune cells. The contradiction is strong in this one. After all, the system...let’s take a look at exosomes. Proficient in transportation, exosomes are tools of communication and provide... (micro RNA) and other molecules that can be transferred to other cells, inducing epigenetic changes in...in targeted cells. In this case, exosomes may transport miRNA from drug-resistant cells to sensitive ones...enzyme that controls apoptosis and regulates transcription. Without TKA1, cells proliferate and cannot ...2009). Then, scientists began focusing on the extracellular matrix and fibroblasts. With each cell mechanism...

...than the traditional SpCas9 enzyme (about a third of the size) and does not require a tracrRNA (it can ... it will proceed to generate a blunt DNA double strand break. Cas9 has been studied for genome engineering...locates its target sequence and initiates single stranded DNA degradation. Cas3 DNA digestion can proceed...convenient for targeting experiments requiring single strand DNA generation, such as homology directed-repair...therapeutic delivery.   Cas14 – Mini, but single strand mighty Unlike many other Cas enzymes, Cas14 targets...it is also significantly smaller in size than traditional Cas9. Another similarity to Cas12a is that Cas14...and cleave RNA through the same mechanism as traditional spCas9. If you are looking to modify RNA, Cas9...

...Adeno-associated viral vectors for functional intravenous gene transfer throughout the non-human primate brain...AAV9-X1.1 efficiently transduce brain endothelial cells across diverse mice strains and rats. Figure from...StayGold variants AAV.CAP-Mac for systemic gene transfer in primate brains Endothelial-specific AAV delivery...electroporation or poor cellular entry with other transfection methods. The Junwei Shi Lab addressed this problem...Editing (PAGE). They fused the Cas enzyme to cell-penetrating peptides and co-incubate it with an “assist peptide...multiplexed guides. Figure 1: A) Cell-penetrating Cas9, fused to HIV TAT, Myc and SV40 Nuclear ...breaching the blood-brain barrier and delivering a transgene, with neuron bias observed in young macaques and...

...growth rate/transfection efficiency is not known, this is easily achieved by splitting transfected cells into...specific cell line, and its transfection efficiency. We often trypsinize the transfected cells on the day after...Many years ago, people in the laboratory became frustrated with using glass cloning rings to isolate colonies... cell lines we most commonly work with, require extra serum to survive at low densities - adding to the...biology-grade agarose containing physiological concentrations of salts and glucose. If we circled well-separated...Agarose cloning method procedure The procedure (illustrated in the YouTube video below) is simple: After ...after transfection into plates containing selection agent at different densities, then dispose of the plates...