...endogenously expressed SCF complex members, an E3 ubiquitin ligase complex. Finally, when auxin is added...can offer over other protein control methods and compare the commonly available types of degron tags. What...removed. The degradation and reversal are rapid compared to other systems because the system modulates ...other systems reviewed here. The only required components are 1) a SMASh-tagged protein and 2) asunaprevir...fusion protein with the endogenous CRBN E3 ligase complex which will polyubiquitinate the fusion protein....target protein-of-interest and the CRBN E3 ligase complex leading to ubiquitination and proteasomal degradation...reviewed here. The AID system relies on three components. First, the protein of interest must be AID-tagged...

...experiment requires delivery of, at minimum, two components: the Cas9 protein and a guide RNA (gRNA) targeting...valuable to scientists, there are some potential complications that must be considered when using this method...alternative approach, which avoids many of these complications, is to directly deliver a ribonucleoprotein ...ribonucleoprotein (RNP), consisting of the Cas9 protein in complex with a targeting gRNA, to your cells of interest. Cas9...cleaving genomic targets with similar efficiency as compared to plasmid-based expression of Cas9/gRNA and can...CRISPR experiment is the generation of the RNP complex.  While one option is to purchase the Cas9 protein... by commercial vendors such as IDT. These two components are then incubated together to form the RNP. ...

.... They are made up of compensation beads and a conjugated antibody. Compensation beads are a mixture between... single color control prior to compensation (B) and after compensation (C) with APC-Cy7.   FMOs FMO...your experiment, and they are non-specific. The compensation beads can bind to any primary antibody you add...up your voltages and can also be helpful for compensation. You should have a single color control for ...They may also be used as a starting point for compensation (Fig. 1B-C). However, it’s important to note...and not experimental samples, the voltages and compensation you set from your single color controls may ... the reference plots provided by the antibody companies or check the literature. Pro tip! Early gates,...

...Delivering CRISPR components to plant cells Once you have selected the correct CRISPR components for your application...deliver these components to your target cells. Remember, efficient delivery of CRISPR components is essential...organisms, some plant-specific changes to CRISPR components are necessary to enable genome editing in plant... academic researchers through Addgene. CRISPR components for plant genome engineering CRISPR can be used...genome of plant cells. The structure of the gRNA (composed of a ~20 nucleotide targeting sequence and ~75...line will result in a failed experiment. CRISPR components can be expressed stably or transiently depending...delivery method and cell type in question. CRISPR components can be delivered and expressed transiently using...

... Note: hypoxanthine (shown) is the nucleobase component of the nucleoside inosine. Created with BioRender.com...and one-ring pyrimidine base. This is a far more complex chemical task, not a job for a single enzyme. ...bind DNA, Cas9 requires (1) a guide RNA, (2) a complementary DNA sequence, and (3) a protospacer-adjacent...removed the uracil glycosylase inhibitor (UGI) components, which inhibit BER during cytosine-to-uracil ...developed two fusions of a CBE with other BER components: the DNA repair protein rXRCC1, or the DNA binding...separate and preventing repair based on the complementary strand. This could explain why the edited U ...original G is not primarily converted back to a complementary C. Optimized base editing with eOPTI-CGBE and...

...approach to monitor the composition of spatiotemporally defined protein complexes. Nature communications...can be part of multiple dynamic and overlapping complexes that have distinct functions. Many existing methods...proteins that interact with a particular protein  complex as opposed to an individual protein. The Bethune... when the bait is part of a particular protein complex. Not performed in a native cell context. For AP-MS...controllable version of the BioID protocol. The critical component for both BioID and Split-BioID is BirA*, a promiscuous...experiment is called a protein fragmentation complementation assay (PCA). In the Split-BioID method, each...another example of a protein fragmentation complementation assay that can be used to study binary protein...

... in proximity by the looping of chromatin, the complex of DNA and proteins that forms chromosomes. These...of a stable cell line that co-expresses three components: dCas9 with an added biotin acceptor site BirA...genomic location of interest  When these three components are co-expressed, the gRNA targets the dCas9 ... of the CAPTURE method. After protein and DNA complexes are cross-linked, DNA is digested into smaller.... This is cumbersome and also makes it hard to compare results across multiple targets because different...with primary cells or rare cell populations. Key components of the CAPTURE 2.0 system To improve upon the... Using this tag reduces the number of CAPTURE components that need to be delivered to cells from three...

...immunoglobulins, to isolate a specific protein out of a complex mix. Using this technique, users can look for the...the antibody or antibody-target protein immune complexes, wash away unbound protein, elute and measure ...Protein extracts from cells or tissues contain a complex mix of proteins seen as green squares, purple circles...protein mix and antibody-target protein immune complexes are allowed to form. Following incubation, beads...beads are used to immobilize the antibody-protein complexes, and as in the first method, unbound proteins ... need to choose the protein type that is most compatible with the host species and isotype of their primary...developed, which can be used for most antibodies compatible with either Protein A or G.  While commonly used...

...point is there are many ways to quantify the composition of a viral vector solution, and generally these...only 20% of recombinant AAV2 particles were full, compared to 50% of wild-type AAV2 stocks (Grimm et al.,...understand what these values mean and how they can be compared.   Figure 2: Label that comes on Addgene...affected by many experimental factors. To list some complicating factors for quantifying AAV: qPCR efficiency...the AAV genome, caused by repeats and self-complementarity of the AAV genome and the ITRs. qPCR efficiency...individual sample, this will reduce convenience and comparability because each sample with be quantified with ...linear region of the assay. This may be due to competition for binding sites between the primers and the...

... plasmids with the same ori are incompatible because they will compete for the same machinery, creating...their copy number, ori, and incompatibility groups. Note the A -C compatibility grouping is an arbitrary ...origin of replication/replicon. The replicon is comprised of the origin of replication (ori) and all of ...origin of replication wisely and avoid plasmid incompatibility In other words, the best choice of ori depends... or not you need to consider your plasmid's compatibility with one or more other plasmids. Generally speaking...plasmids for an experiment, make sure they have "compatible" ori's. See the table below for more details....details.   Common Vectors Copy Number+ ori Incompatibility Group Control pUC ~500-700 pMB1 (derivative...

...But what exactly is an antibody, and how is a component of the immune system useful as a research reagent...heavy chains, and the rest of the Fab regions are completed with the Ig light chains. The variable region ...production strategy has advantages and disadvantages compared to each other. Polyclonal antibodies These antibodies...Single-chain variable fragment (ScFv) Figure 2: Comparison between the IgG antibody and scFv. Because... their lower affinity for their target protein compared to whole antibodies (Crivianu-Gaita 2016). Get...Variables (scFvs). Nanobodies Figure 3: Comparison of the Hcab and nanobody. Nanobodies are the...fluorescence. Western blots are frequently used to compare relative levels of protein expression between cell...

...gene-of-interest, all the way to the complex multi-TU constructs that compose genetic circuits and metabolic... regulators, and lots of viral components. This kit is also compatible with the Joint Modular Cloning ...noticed that studies frequently made comparisons between CRISPR components expressed from different plasmid...extensive with a supplemental add-on kit and is compatible with E. coli and/or cell-free systems. Protein... backgrounds, MoClo can be invaluable for more complex editing and screening experiments in organisms ...nature of the Fragmid system allows for direct comparisons of CRISPR parts. The Fragmid system was used ...plasmid backbone elements impact viral titer and to compare different Cas enzymes in all-in-one AAV delivery...

...effects, ABEs also compare favorably to other methods. In a head-to-head comparison with Cas9, Cas9 modified...double-strand breaks (DSBs) preferred by researchers. Compared to non-homologous end joining (NHEJ), HDR occurs... Note: hypoxanthine (shown) is the nucleobase component of the nucleoside inosine. Created with BioRender.com...represent the fourth generation of base editors. Compared to BE3, BE4 offers a 2.3 fold decrease in C→G ...frequency of 1.2%. Although the lab did not conduct comprehensive genome-wide studies of ABE specificity, their... 40 new ABE8 variants (Gaudelli et al., 2020). Compared to ABE7.10, ABE8s resulted in 1.5-fold more editing...higher editing efficiency at non-NGG PAM variants compared to ABE7.10. ABE8s have an improved base editing...

...many opportunities to learn from other biotech companies at workshops and can use what we learn to improve...office space shared by numerous emerging biotech companies. The space was modern with clean bold colors of...hallway for people to take a break and relax. Companies at LabCentral get access to a variety of experimental...how LabCentral aims to enable emerging biotech companies, I was excited to go there because both LabCentral...must first fragment a DNA sample into many small components. Without proper processing, these small DNA strands... workshop participants from different biotech companies to interact and learn more about one another. ...expand my skills and network with other technicians/company representatives. I got to talk with a technician...

...recently developed a photosensitizer composed of the protein complex complex and the MG-2I-dL5** fluorogen that...thus forming the full FAP-TAP complex (MG-2I-dL5**).   A FAP-TAP complex will light up if excited with...cells. The photosensitizer used in this work is composed of two parts: 1) an activatable protein (the FAP...-TAP MG-2I-dL5** MG-2I-dL5** is a new FAP-TAP complex where dL5** is the FAP. It’s job is to bind MG-...fluorogenic dye.  MG-2I is the TAP portion of the complex and is derived from the malachite green (MG) dye...modified with the addition of two iodine residues. Compared to MG, MG-2I has increased ROS generation when...with these fluorescent proteins. He et al also compared MG-2I-dL5** head-to-head with KillerRed, a genetically...

...consistent, and controllable pulses of light. Complicated patterns of light intensity and wavelength can...not E. coli. They fall under the heading of two-component systems (TCSs). TCSs consist of a histidine kinase...created the first E. coli light-sensitive two-component system (TCS). Levskaya et al. fused a photosensory...dark conditions, the chromophore activates the RR (OmpR) via phosphorylation, promoting transcription (see... image, as well as a genetic method for the computational problem of edge detection. Tabor et al. subsequently...gene expression. An E. coli light-sensitive two-component system (TCS). A hybrid histidine kinase/photosensor...were spread over a number of plasmids, with one RR (ompR) encoded chromosomally. Leakiness was also an issue...

...that the classroom cannot offer. Furthermore, companies across most industries tend to hire their former...Prior to March 2020, internships were a fiercely competitive endeavor. Due to the COVID-19 pandemic, students...college, and graduate students. In order to gain a competitive advantage this cycle, consider the advice below...which is the perfect opportunity to paint a comprehensive picture of your academic interests, potential...potential, and drive. Read the associated prompt carefully, plan your response, and let your inner wordsmith... impress the judges on your ability to tell a compelling narrative that is positive, and forward-looking...counterintuitive since that approach seems to create competition for your own spot. True! But you create a network...

...isopropyl β-D-1-thiogalactopyranoside). (To be completely accurate, IPTG binds and inactivates the lac ...to a lacZΔM15-mutant bacterial cell in trans complements the mutation allowing for a functional enzyme...enzyme. This process is called α-complementation. The system described above was put to practical use in the... inserted it into a plasmid, creating an α-complementation cloning vector. When a cloning reaction goes...interrupted as shown in cell A, and thus will not complement the cell's β-galactosidase mutation. An unsuccessful...functional β-galactosidase enzyme through α-complementation (cell B). As shown on the representative plate... use a proper plasmid (i.e. contains the α-complementation cloning MCS): pGEM-T, pUC18 and pUC19, and ...

...cerevisiae yes - This can complement ura4- S. pombe, but the complementation is weak. yes   LYS2 L-lysine...vector propogation. This post, along with a future companion post on mammalian vectors, will catch you up on...vectors? Yeast are eukaryotes and thus contain complex internal cell structures similar to those of plants...in the lab typically contain all the necessary components to allow shuttling between E. coli and yeast ...an organism to synthesize a particular organic compound required for its growth. Many auxotrophic strains... including a copy of a functional gene which complements the host’s auxotrophy. When grown on media NOT...LEU2 L-leucine no S. cerevisiae yes - This can complement leu1- S. pombe, but mulitple copies are required...

...of the protein MLH1, a component of the MutSα–MutLα mismatch repair (MMR) complex, to temporarily inhibit...45-fold) higher across different genomic sites compared to PE1 (Anzalone et al., 2019). PE3: Resolving...recognized as an MMR intermediate, and the MutSα–MutLα complex may either selectively excise the edited DNA flap...the unedited strand. Temporarily inhibiting this complex may allow time for 5′ flap exonucleases and DNA...nuclease activity (Chen et al., 2021). In a system as complex as prime editing, every efficiency counts! The ...deliver with AAVs but still efficient at long and complex edits. The team also developed PE6e-g editors with... the truncated pegRNA can still guide Cas9 and compete with full pegRNAs for access to the target site...