...occurring E. coli plasmid ColE1 (Goebel & Helinski, 1968; Bazaral & Helinski, 1968), in which the plasmid was ...1981). E. coli strains such as DH5α, HB101, NEB Stable, Stbl3, etc., are recA- mutant strains, and therefore... of plasmids, taking many forms (Levene, 2009; Higgins & Vologodskii, 2016). Plasmid multimerization occurs...to occur more frequently in plasmids with large inserts (>7 kb)(Berg et al., 1989), direct repeats (Ribeiro...et al., 2009). Since multimers have additional origins of replication compared to monomers, they are also... of multimer present in your sample. In these instances, please note that the supercoiled dimer band can.... If the presence of a multimer in your sample remains unclear after visualizing it on a gel, one of the...

...Mutations Insertions, deletions, point mutations Insertions, deletions, point mutations Insertions, deletions...-4 phage proteins are needed and these can be provided via a separate plasmid or be inserted into the ...recombineering proteins Red/ET from a temperature-sensitive plasmid. Homologous recombination then joins the linear...successfully incorporated a 60-nucleotide insertion; beyond this insertion size, primer synthesis is more expensive...making small point mutations, large deletions/insertions require complicated, often costly in vitro assembly...the power of bacterial recombineering to create insertions, deletions, and substitutions - at the same efficiency...colonies carrying the unmodified plasmid. For insertions, it’s possible to use the same technique just...

...correct insert (gRNA) in pX330 / pX335 with BbsI/AgeI double digestion. As a successful insertion will destroy... have been published with thorough step-wise instructions on designing and using Cas9-based genome engineering...vector (like PX330). For large changes (>100bp insertions or deletions), we typically use plasmid donor...homology arms on each side flanking your desired insertion or mutation. Each arms is around 800bp. For transfection...CRISPR/Cas9 system to create specific mutations or insertions by Homologous Recombination (HR), what is the...purified long oligos. For large changes (>100bp insertions or deletions), the Zhang lab typically uses a...homology arms on each side flanking your desired insertion or mutation. Each arm is around 800bp. For transfection...

...REC and NUC, and their subdomains. NUC includes the HNH and RuvC catalytic domains, as well as the CTD (...recognition). The NUC lobe contains the DNA scissors: the HNH and RuvC domains, which are named for similar...the guide RNA. Unlike the cleavage domains, the recognition domains—including the CTD and the entire REC...similar nuclease folds found in other proteins. The C-terminal domain (CTD) of the protein also belongs ...lobe—do not structurally resemble any other known proteins; they are completely unique to the CRISPR system...Nishimasu et al., 2014). Table 1. Summary of Cas9 domains, the lobe each domain belongs to, and their roles...pyogenes Cas9 in the apo state (PDB ID 4CMP) with domains colored as in Figure 1. HNH, red; RuvC, orange;...

...binding of untagged proteins. Despite being a relatively low frequency amino acid, proteins containing two ...research requires a close examination of specific proteins within a system. This can be pretty complicated...given that a single cell has tens of thousands of proteins functioning in a variety of ways. How do scientists...from a complex mix using affinity chromatography against an amino acid sequence, or tag, on the protein....of methods can be used to successfully isolate proteins, this blog will focus on one of the most common... as Ni2+ or Co2+ (Hochuli 1988), and can elute proteins with up to 95% purity (Janknecht 1991, Hochuli...itself. Next, a series of wash steps removes excess proteins and other contaminants. The protein of interest...

...Mullineaux Insulin Sensor for Imaging Insulin Secretion A Ratiometric Sensor for Imaging Insulin Secretion... Collections Fluorescent Proteins Fluorescent Biosensors Fluorescent Proteins: Biosensors Metal Ions Small...Service: AAV Biosensors Fluorescent biosensors are proteins designed so that their fluorescent properties ...transfer (FRET) efficiency of a pair of fluorescent proteins. Genetically encoded biosensors can be introduced...Constitutive or Cre-dependent) Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature. 2013 Jul...nested reporters of green- and orange-emitting proteins Ratiometric Matryoshka biosensors from a nested...cassette of green- and orange-emitting fluorescent proteins. Nat Commun. 2017 Sep 5;8(1):431. Wolf Frommer...

... and analysing the insecticidal potential of Cry proteins in order to manage insect pests and to support...The recombinant Cry proteins will then be screened for toxicity against local insect pests. By developing...the identification of plant compounds and insecticidal proteins to the development of renewable biotechnology...desaturating fatty acids. Identification of Insecticidal Proteins Linh Bao Ton (M. Sc.), Nong Lam University...developing biological agents such as insecticidal proteins, the use of chemical pesticides may be avoided or...system pDawn to clone and express newly identified proteins and enzymes. These compounds will later get tested... nutritionally and therapeutically significant proteins. In this project she aims to create transgenic...

... EF1a-tTA-2 Contains Gateway L1L3 sites and EF1α promoter driving tTA; contains insulator sequence; plasmid...Blasticidin selection. Please NOTE, this vector contains an insert (MAT2A) which would need to be replaced by...Backbones Find a construct that will allow you to insert and induce your gene of interest. ID Plasmid Description...format: PGK-rtTA-2A-puro; see article for tagged insert options rtTA On Root 11651 pLVUT-tTR-KRAB Lentiviral...bidirectional tet-responsive promoter (Pbi); Pbi contains a TRE between two minimal CMV and is silent in...third-generation rtTA On Elledge 19407 pTREtight2 Promoter contains a modified TRE that is silent in absence of binding...

... of proteins captured on magnetic beads (Arimura et al., 2024). In MagIC-cryo-EM, target proteins (expressed...adding Late Embryogenesis Abundant (LEA) proteins, small proteins that naturally adsorb to the AWI, can ...editor installs the recombinase “landing sites,” then the recombinase handles the installation of the ... DNA-targeting proteins, so the team created versions using dCas9, zinc finger proteins, and transcription...for enrichment and structure determination LEA proteins protect samples during cryo-EM grid freezing Engineered...biotinylated SpyTag-SpyCatcher linkers and spacer proteins ending with a GFP nanobody (Figure 1). This enrichment...https://doi.org/10.1101/2024.01.21.576499.    LEA proteins protect fragile samples during cryo-EM grid plunge...

...Broad Institute focuses on proteomics: developing and applying new technologies to quantify proteins in ...locus and labels the proteins in its vicinity with biotin. The biotin-labeled proteins can then be isolated...electron-rich amino acid side chains (e.g. Tyrosine) with biotin. These biotin-labeled proteins can them be analyzed... anti-GFP nanobody is able to bind fluorescent proteins derived from A. victoria the functionalized nanobodies...: 29915061.        A new method to identify proteins occupying a specific genomic locus: Caspex  Article...group, developed a CRISPR-based approach to detect proteins that associate with specific genomic loci. He ...engineered peroxidase, (APEX) that labels proximal proteins with biotin as an affinity tag. This ‘Caspex’ ...

... Chavez, researchers at the Wyss Institute for Biologically Inspired Engineering. CRISPR/Cas9 technology...to effector domains such as transcriptional activators or repressors. These fusion proteins retain the .... For instance, when native (nuclease-competent) Cas9 interacts with a ≤15nt gRNA that contains an RNA..., or guide RNA (gRNA). The gRNA conventionally contains a 20-nucleotide sequence that is complementary..., in the genome. Native Cas9 has two catalytic domains, each of which cleaves one strand of DNA upon binding... full-length gRNA, native Cas9's two catalytic domains, HNH and RuvC, cleave DNA strands complementary... a regulator of gene expression. Its catalytic domains can be mutated to inactivate the protein’s nucleolytic...

... measurement that is unaffected by intervening proteins, lipid bilayers, organelles or other cellular ...et al., 2007). All that is required is that the proteins/nucleic acids/lipids of interest are fluorophore...resulting from nonspecific donor labeling. Fluorescent proteins (FPs) are the gold standard for genetically-encoded...remember that, if you make an FP fusion, you are inserting about 20-25 kDa of protein mass into your protein...protein and so structural changes due to this bulky insertion are inevitable. Most FP fusions for FRET studies...can affect the structure or function of certain proteins. Rather than shove a bulky FP into your protein...pair, a researcher can lure the ribosome into inserting a fluorophore-tagged amino acid at a stop codon...

... Vector Database. Step 2: Insert name  Include information about the insert in your plasmid name. This...position to the insert. Example, if you have a Flag tag on the N-terminal of your insert, you would list...fused to the C-terminal of your insert you would list it after the insert. pBACKBONE-Flag-hGene-EGFP Other...Other considerations  If your insert contains a mutation or modification, this should be included in the...versions of the insert are often denoted by "wt" either directly before or directly after the insert name.   ... quickly and easily identify what your plasmid contains and other important information. Tip: A lowercase...antibiotic resistance, b) the promoter that drives the insert, and c) any other selection markers (for use in...

...Plasmid Gene/Insert Promoter Selectable Marker PI Publication Bacteria ID Plasmid Gene/Insert Promoter PI...Plasmid Gene/Insert Promoter Selectable Marker PI Publication Plant ID Plasmid Gene/Insert Promoter Selectable...Plasmid Gene/Insert Promoter Selectable Marker PI Publication Yeast ID Plasmid Gene/Insert Promoter Selectable...Plasmid Gene/Insert Promoter Selectable Marker PI Publication Xenopus ID Plasmid Gene/Insert Promoter Selectable...joining (NHEJ), a mechanism which frequently causes insertions or deletions (indels) in the DNA. Indels often...repair a DSB using homology-directed repair (HDR) instead of NHEJ. In most experimental systems, HDR occurs...Marker PI Publication Parasites ID Plasmid Gene/Insert Promoter Selectable Marker PI Publication Do you...

... Plasmid Collections Fluorescent Proteins FRET Fluorescent Protein Guide: FRET More Fluorescent...fluorescent. A variety of organic dyes and fluorescent proteins have been developed for use as donor and acceptor...intramolecular or unimolecular FRET). Fluorescent proteins can be used as donor and/or acceptor molecules...suitable for creating individual fluorescently tagged proteins to study protein-protein interactions and a series...laboratory. Empty Vectors Encoding Fluorescent Proteins for FRET The following plasmids can be used to...brightness standard consisting of two Venus fluorescent proteins connected via a 5 amino acid linker V17V Anisotropy...brightness standard consisting of two Venus fluorescent proteins connected via a 17 amino acid linker V32V Anisotropy...

...Zhou: Fluorescent Light-inducible Proteins (FLiPs) Fluorescent proteins can be applied in a long list of...light. To do so, she generated fusion proteins in which Dronpa domains flanked either side of an enzyme’s...Fluorescent proteins have enabled scientists to pursue creative research avenues previously unavailable...researchers are finding new ways to apply fluorescent proteins everyday.  The late Michael Davidson and Roger...for their creativity in the use of fluorescent proteins and the potential for their work to enable future...that list by using photoswitchable fluorescent proteins to create enzymes that can be turned on and off... exposure caused oligomerization of the Dronpa domains thereby occluding the enzyme active site and shutting...

...for tDNA insertions into the plant’s germline Selecting for seeds that have the tDNA insertions (usually...have no control over how many insertions occurred. Having too many insertions can cause artifacts from over-expression...control over where your construct will be inserted. If your insertion occurs in a coding region, it may cause...transcriptional and translational dynamics of genes and proteins in living plants, to complementing mutant phenotypes... process occurs in nature; Agrobacterium tDNA insertion likely precipitated domestication of sweet potatoes...and generating N and C terminally tagged fusion proteins with fluorescent (YFP, GFP, CFP, RFP, etc.) or...perform the transformation not long after flowering begins. If your plants begin to flower before your constructs...

...only indicates the presence of AN insert, not necessarily YOUR insert. Any cloning artifact that disrupts...able to choose those that contain plasmid with an insert? Blue-white selection is a widely used method to...beginning. The well-characterized bacterial lac operon contains a gene called lacZ that encodes for the enzyme...indolyl-β-D-galacto-pyranoside) into galactose and an insoluble blue pigment (4-chloro-3-brom-indigo). So far,...an orange wedge in the figure on the left) and inserted it into a plasmid, creating an α-complementation...representative plate to the right, colonies with an insert-containing plasmid have a non-functional β-galactosidase...control: Transform the backbone plasmid without insert. All colonies on this plate should be blue, indicating...

...Microbial rhodopsins are encoded by opsin genes, and include bacteriorhodopsins, halorhodopsins, and channelrhodopsins...of optogenetics begins almost 50 years ago with the discovery of microbial rhodopsins, light-sensitive...light-sensitive proteins that absorb photons for energy conversion or the induction of intra- or intercellular signaling... et al., 1984; Harz and Hegemann, 1991). These proteins control the flow of electric charges across cell...light (Zhang et al., 2011). What do microbial rhodopsins and neurons have in common? Neuronal stimulations...when hit with the correct wavelength, microbial rhodopsins enable ions to flow across the axon’s membrane... 2012). For example, blue light activates channelrhodopsins that, in turn, trigger neural excitation, ...

...system is likely applicable to many types of proteins. For proteins with a modular structure, constructing ...Other proteins may also be utliized as scaffolds, including the set of red fluorescent proteins. The T-DOGGs...researcher Jonathan Tang wanted to express a variety of proteins in single cell types in the mouse, but knew it...single heavy chain, and they are small and stable inside cells. Nanobodies binding GFP were designed in ...showed that nanobodies could be fused to other proteins, and these fusions retained the ability to bind... Tang et al. tested pairs of their GFP-binding proteins (GBPs) to find those that could co-occupy GFP....similarly to GFP, but commonly used red fluorescent proteins dsRed, mCherry, and TdTomato do not induce transcription...