...model organism, they insert into the host’s genome. Then, the transposon insertion sites are detected with...transposase proteins (TnsA, TnsB, TnsC), and 4) the donor DNA or mini-transposon that contains the DNA cargo...and result in human disease. While transposon insertions can be detrimental, it also drives genomic evolution...that moves around. This DNA sequence encodes the proteins the transposon needs for transposition. Transposition...transposition.  Target site: Different transposons insert at different DNA sequences or target sites. Integration...duplication of this target site sequence at the site of insertion.  Types of transposons Transposable elements ... reverse transcribe back into DNA before being inserted into new target sites. This is similar to how ...

...fluorescent proteins that are photostable. On top of photobleaching, fluorescent proteins may display ...performed with soluble fluorescent proteins or localized fusion proteins and do not require dedicated plasmids...performed on purified fluorescent proteins. To avoid diffusion, the proteins are (i) trapped in microdroplets...course of the experiment. The best fluorescent proteins for live cell imaging can be excited many times...fluorescent protein. This can differ between fluorescent proteins in the same spectral class (Cranfill et al, 2016...confocal laser scanning microscopy, fluorescent proteins are excited with very intense light (for a brief...compare the photostability of different fluorescent proteins? Measuring photostability Since it is clear that...

...   Dye stains Dye stains are non-antibody-based stains that bind DNA or free amines of proteins. Depending...cytosol/nucleus, or visualizing DNA instead of proteins. Some stains can diffuse through the cell membrane...transmembrane proteins, such as receptors, or post-translational modifications on those proteins, like glycosylation...kits allow for labeling of cytosolic proteins, as well as proteins of the secretory pathway. Those can ...buffer to label intranuclear proteins of interest, you can label cytosolic proteins alongside the intranuclear...like many of the cluster of differentiation (CD) proteins found on immune cells. However, there are more...methods can include labeling markers with multiple proteins/antibodies, targeting markers found within the...

...phosphoserine-incorporated proteins The generation of pSer-incorporated proteins requires a molecular toolkit... Sep-OTS, contains the components required for the translation of pSer-incorporated proteins on a single...Niemi, a postdoctoral fellow at the Morgridge Institute for Research in Madison, Wisconsin. It is commonly...cited that approximately one-third of cellular proteins are modified through phosphorylation (1). However...of a protein for characterization. However, phosphoproteins are often low abundance, and thus difficult...incorporation of phosphoserine directly into recombinant proteins (5). Optimization of this system defined three...effectively incorporate phosphoserine into recombinant proteins, such as the kinase MEK1 (5). MEK1 is inactive...

...generate two more OptoNBs, one against mCherry and one against GFP. Insertion of AsLOV2 into loop 1 always...how AsLOV2 insertion into mCherry or GFP nanobodies affected binding of their target proteins. The surface-exposed...opposite effects: insertion into loop 1 caused dark-induced binding while loop 6 insertion caused blue-light... you wash several times to remove non-specific proteins, and then elute YFP.  Option #2: Opto-Nanobodies... purification You skip adding a tag to YFP and instead use OptoNBs. You fill a column with OptoNB coated... the lights, OptoNBs bind YFP and non-specific proteins flow through. To elute YFP, you turn on the blue...ability of its nanobody partner, so it’s important to insert AsLOV2 at a spot in the nanobody that preserves...

...scientists have used fluorescent proteins to observe what’s happening inside cells for at least a couple of...earlier described. “Until our proteins, there were lots of fluorescent proteins made from jellyfishes and ...decades. Green is the classic color, but fluorescent proteins are available in a variety of hues. While those... applications, Verkhusha and his lab at Albert Einstein College of Medicine in New York recognized their... The key to their bacterial phytochome-derived proteins, iRFP670, iRFP682, iRFP702, iRFP713 and iRFP720... The challenge then was to develop fluorescent proteins that would fall within that near-infrared range... and that’s exactly what iRFPs achieved. Those proteins allowed a signal-to-background ratio in mammalian...

...Rep78 and Rep68 contains a DNA-binding and endonuclease domains, all four Rep proteins share a common ...which contains the AAV structural and packaging genes, the adenoviral helper plasmid which contains the ...the proteins needed for the virus to replicate,  and the transfer plasmid which contains the viral genome...Rep proteins. Alternative splicing generates the different C-terminal regions of the Rep proteins. While...common helicase and ATPase domains. To function, the Rep proteins form oligomers of Rep 78/68 or Rep52/40. ... start sites generate the three VP proteins. The three VP proteins assemble into capsid at a 1:1:10 ratio...gene of interest can be packaged into the virus instead.  Next let’s take a closer look at the rep and ...

..., the red fluorescent proteins form tetramers. In general, fluorescent proteins have a natural affinity...true monomeric fluorescent proteins. Concluding remarks Many fluorescent proteins are available, and their...aggregation The tendency of purified fluorescent proteins to form homodimers or higher order oligomers can...-PAGE under non-denaturing conditions separate proteins based on size. Whether higher molecular weight...protein, reflect the likelihood that fluorescent proteins interact or oligomerize in cells? This question...question is largely unanswered. Ideally, fluorescent proteins should act as inert modules that fail to homodimerize...homodimerize in cells. To understand how well fluorescent proteins approach this ideal situation, cellular assays...

...bacterial genome, and inserted it into E. coli to express and then test for any proteins that could cut RNA...the lab instead turned to phage serine integrases, where they saw 10-20% integration of inserts up to 36...Schematic of programmable gene insertion with PASTE. The PASTEsystem involves insertion of landing sites via Cas9...single-stranded nicks instead of the double-stranded breaks used in other insertion systems. Off-target ...nonetheless a new subtype. Cas7-11 had combined all the domains into a single protein, shedding the complexity ...Cas7-11   Use The Cas7-11 targeting system contains two pieces: a single protein, computationally ...computationally described as a fusion of four Cas7 proteins and a putative Cas11-like protein, and a guide RNA. The ...

...editing techniques. Inbred and outbred strains Mouse strains used in research can fall under one of two.... Common inbred strains include C57BL/6, Balb/c, and 129. Conversely, outbred strains were originally ...Examples of outbred strains include Swiss Webster and Harlan (Hsd) National Institutes of Health (NIH) Swiss...broad classifications: inbred or outbred. Inbred strains share a common genetic background, which means ...limited but genetically diverse gene pool. Outbred strains are strategically bred to maintain maximum genetic...genetic diversity of the human population. Outbred strains of mice are useful for comparing dominant and recessive...Genetically engineered mice Mice can be engineered by inserting a targeting vector containing the gene of interest...

...Hospital  Brandeis University  Broad Institute  California Institute of Technology Cold Spring Harbor Laboratory...Laboratory  Dana-Farber Cancer Institute  Fred Hutchinson Cancer Research Center Memorial Sloan-Kettering...  National Institute of Diabetes & Digestive & Kidney Diseases (NIDDK)  National Institute of Genetics...Rockefeller University  Salk Institute for Biological Studies The Scripps Research Institute St. Jude Children's...University and Medical Center  Weizmann Institute of Science Whitehead Institute for Biomedical Research Glossary...intellectual property, and to help requesting institutions safeguard themselves from undue liability. Before...materials? If so, you should always talk to your institution’s TTO or legal office first. Generally, Addgene...

...could be affected by the location of insertion. Lentiviral insertion can also disrupt the expression of ...Low Efficient and Stable Genome integration Insertional mutagenesis, transgene reactivation, residual...Low Efficient and Stable Genome integration Insertional mutagenesis, residual expression piggyBac ... inefficient Difficult to purify reprogramming proteins. Table 1: Key Features of Different Methods...of iPSC generating methods due to the risk of insertional mutagenesis. Random integration also creates ...potential. Finally, retroviruses are prone to insertional mutagenesis and random integration creates variation...Addgene. Another drawback is that lentiviral vectors insert in different genetic locations in each transduced...

...generations of scFvs against two antigens that are impossible to generate scFvs against in vivo: antigens...the sequences for the variable domains of the selected scFvs are inserted into a vector encoding the antibody... are often the building blocks for engineering proteins. So let’s get acquainted with scFvs. What is an...variable heavy chain (VH) of an antibody. These two chains are connected by a flexible linker peptide that...scFv. While you might assume that the variable domains of an scFv would mirror that of an antibody (VL-linker-VH...antigens found in the body or self antigens, and toxins that are lethal to animals.  In the clinic, scFvs’ size...to produce scFvs in mammalian, yeast, plant, and insect cells, they are most often expressed from a plasmid...

...ankyrin repeat proteins, or DARPins, are genetically engineered small affinity proteins, around 14-15 ...antibodies. Instead, they are derived from a class of binding proteins known as ankyrin repeat proteins. DARPins...and heavy chains made by immune systems. But here, we're not interested in antibodies. Instead, we're going...engineered fusion proteins similar to a Fab fragment but created through engineering instead of cleavage. ...synthetic binding proteins which use the Fibronectin type III domain as their scaffolding instead of the antibody...the ability to specifically recognize and bind proteins. They’re used in a number of research and clinical...these alternative reagents are created in the lab, instead of generated in vivo, they have higher reproducibility...

...  The best applications for dsDNA inserts include: large insertions or deletions, including selectable...note* not all linked strains contain the Lambda Red system).  In some of these strains, expression of exo... used to make an assortment of modifications: insertion and deletion of selectable and non-selectable ...of the experiment. dsDNA substrate is best for insertions or deletions greater than approximately 20 nucleotides... only a few base pairs.  dsDNA Substrate dsDNA inserts can be made by PCR using primers that amplify the... with 50 base pairs of homology to the targeted insert site (Figure 2, top).  These primers are typically...not determine the direction of replication and instead design oligos that target both strands. One of ...

...high quality DNA that contains these elements. Be aware of endA+ strains Strains like Stbl3 have been ...incomplete. Protein expression strains are not good for cloning Bacterial strains adapted for protein purification...bacterial strain Differences between E. coli host strains can impact plasmid yield. Check the bacterial strain...an example). Look out for carbohydrates Certain strains, including HB101 and derivatives, release large...have previously reviewed many common lab E. coli strains on the blog, and also recommend this handy guide...for a more extensive listing of common E. coli strains. Increase the volume Starting with a larger culture... be affected by the plasmid size, nature of the insert, propagation strain, growth conditions, and the...

...tags and fusion proteins to aid in the expression and purification of recombinant proteins. Tags for stability...recombinant proteins for one-step purification with glutathione, can also protect against proteolysis....one form of instability. Prokaryotes can also have a hard time folding eukaryotic proteins. You can get...101 has covered GFP and its related fluorescent proteins, which are sometimes used as tags for detection... biologists who need to overexpress and purify proteins can face any number of technical challenges depending...protein (MBP). Overexpression can also lead to insolubility, and aggregated protein is not useful protein... but scientists may also choose to add smaller proteins, such as Thioredoxin A (TrxA) that improve disulfide...

... of posts explains how to critically assess the reported properties of fluorescent proteins, how to do...task. Many fluorescent proteins are available and the number of fluorescent proteins is steadily increasing...Before you start a new project with fluorescent proteins, the best advice is to try a couple of promising...do a head-to-head comparison of fluorescent proteins and how to make a well-informed decision on the best...data that summarizes properties of fluorescent proteins on websites. To make the best choice, the fluorescent...relevant conditions. The properties of fluorescent proteins are usually only examined under a few conditions...and provides a quick way to compare fluorescent proteins, e.g. see Chudakov et al (2010), Cranfill et al...

...working with purified proteins in vitro, you can even use the same tag for multiple proteins but label each ..., V. W. (2011). Chemical Tags for Labeling Proteins Inside Living Cells. Accounts of Chemical Research...protein of interest tagged with different fusion proteins for different experiments over the years — a green...been mutated to block its catalytic activity and instead become trapped in a covalent bond with its substrate... dyes provide many advantages over fluorescent proteins, opening up new applications.   Figure ...Griffin et al., 1998), this 15 amino acid peptide contains a cluster of four cysteines (CCXXCC, where X is... Michael Sheetz labs demonstrated in 2005 that proteins fused with E. coli dihydrofolate reductase (eDHFR...

... yeast overexpression vector. This plasmid contains origins of replication and selectable markers that...the S. cerevisiae met deletion strains and then isolate transformed strains on media that lack uracil, but...function wide open. What if two seemingly similar proteins have acquired divergent functions due to mutations...research questions. The idea is straightforward. Instructors first select a multi-gene pathway to study and...and a reference organism where deletion strains are available. Then, staff or students clone similar sequences...overexpression plasmids. Students next transform deletion strains from the reference organisms with the overexpression...addition, a complete set of well-annotated deletion strains is available (6). Methionine synthesis in S. cerevisiae...