...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...

...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...

...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...

... 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...

...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...

...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 propagation. 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 multiple 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...

...projects in introductory labs. Genome projects use computational methods to identify genes by their similarities...abnormalities due to the deletion (i.e. the plasmid complements the deletion). If the phenotype is corrected,..., well-studied and inexpensive. In addition, a complete set of well-annotated deletion strains is available... the absence of methionine (i.e. the plasmid complements the deletion). Find the Plasmids Here Plasmid...blotting in other experiments (Figure 2).   Complementation reveals conservation of function Students have...Addgene, to successfully analyze Met protein complementation. In the procedure diagrammed in Figure 3, students...promoter controls MET/Met gene expression, complementation should only occur when galactose replaces glucose...

... for a new generation of ABEs), consists of 3 components:  a deaminase (TadA, originally from E.coli,... or Cas nickase)  a guide RNA that targets the complex of TadA and dCas to the genomic DNA of interest...effect of removing the wild-type TadA from the complex the researchers developed a second set: The ABE8...resulted altogether in 40 new ABE8 variants. When comparing on-target DNA editing efficiency in a human cell...found that the smaller version, ABE8.x-m, showed comparable editing behavior to ABE8.x-d. Overall, ABE8s ...showed a median increase of 1.94x editing activity compared to ABE7.10. Depending on the position in the protospacer...line while reducing indel frequency more than 90% compared to ABE7.10. Choosing Cas9 proteins to increase...

... human body is made up of organs, cells comprise compartments and structures, called organelles. Take ... the subcellular localization and composition of cellular complexes are important steps in understanding...that the fusion protein localizes properly by comparing the signal from the fusion itself to the wild-...and potentially which other proteins it forms complexes with. These “colocalization” studies give insights...within the same subcellular structure or protein complex. By coexpressing both the marker protein and the...possible to determine the location of proteins within complex structures. In order to interpret colocalization...quantifiable results and statistics that can be used to compare between different experimental setups. A colocalization...

...contains a composite of GenBank, EMBL, DDBJ, and PDB sequences and may be the most comprehensive for searching... Tool (BLAST) provided by NCBI. BLAST helps us compare the sequencing results of the plasmids in our repository...deletions, or insertions. We use blastp or blastx to compare our sequencing results to protein sequences to ...page, the first decision to make is whether to compare a Sanger sequencing result to a single known reference...BLAST search to perform and will save you time compared to other types of BLAST searches. If you do not...megablast, but can return longer alignments to compare with your sequencing trace file. Unlike megablast...consistently run our blastx searches with the “Low complexity regions” filter unchecked so that these regions...

... most successful computer languages are free to use. Of the 15 most popular computer programming languages...engineered DNA systems capable of storing data and computing inside living cells. He's also a co-founder of...digital genetic switches that enable cellular computing. These logic gates built from transcriptors are...BPA) Plasmid Collection along with a BIOFAB kit comprised of well-characterized bacterial transcription .... What the BioBricks Foundation is seeking to accomplish is to make the engineering of biology much easier...carefully engineered to support reliable functional composition. In other words, these parts can be used over...logic shows up in mechanical systems, plumbing, computers, and now through BIL gates we've implemented them...

... as the building block of our understanding of complex biological processes. Many tools exist to perturb...silencing,” of any gene that contains sequence complementary to the dsRNA (Wilson and Doudna, 2013). RNAi... translation, or chromatin modifications of complementary gene sequences (Figure 1). As a result, gene...unmodified dsRNA elicits immunogenic reactions that complicate the effectiveness and interpretation of RNAi.... of interfering RNAs.   RNAi vs. CRISPR: Complementary techniques It is fair to say that CRISPR has...Stainier, 2020). Additionally, a study that directly compared CRISPR to RNAi in the context of a high throughput...data generated from both methods did a more comprehensive gene list emerge that accurately reflected the...

...scientific art competitions where creations can go on display for all to enjoy. The largest competition of this... University’s Annual Art in Science competition. This competition has gained so much public interest for...moonlighting as artists can compete in the Nikon Small World Photomicrography Competition - a 40 year old international...voyage. Previous destructive techniques were incompatible with the need to conserve these unique samples...been featured in Time magazine . Other smaller competitions are now taking place and gathering momentum,...international competition that last year received over 2,000 entries. Do you mix your passion for art and...

...them for use in your lab. Structural Comparison Functional Comparison Advantages of Nanobodies How to Use...for a comparison of nanobody, HCab, and traditional IgG antibody structures. Functional comparison Strong...nanobodies and how their structure and function compare to IgG antibodies, as well as how to produce them...Nanobody Toolbox Find Nanobody Plasmids Structural comparison As the name suggests, nanobodies are smaller ...incubated together without forming large oligomeric complexes. Antibodies are also too large to be expressed... antibody incubation. See figure 4A below for comparison of this one-step staining with nanobodies vs ...localization patterns as cells stained for one target (compare nanobodies staining in figure 4A vs 4B). How to...

... function as a kind of “GPS for the genome” to compute coordinates for genetic elements like protein, ...action at a distance.” Through the marvel of DNA compaction, an enhancer can increase the expression of a...millions of base pairs away. (For details on DNA compaction and the structural proteins that make it possible...appendix DNA is a 3D fractal). Enhancer biology is a complex and dynamic field. We’re going to focus on a tried...details. DNA is a 3D Fractal Figure 2: DNA compaction. Graphics courtesy of beyondthedish (left) and...People are wrong. In reality, cellular DNA is a complex three-dimensional globule of chromatin. Chromatin...tertiary structures (globules) to achieve exquisite compaction—on the order of 700 terabytes per gram—and form...

...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...

... advise pharmaceutical companies, biotech start-ups, health insurance companies, and governments on the...the part of the company involved: Research & Development (R&D) teams at pharma companies carry out preclinical... acquisitions of or collaborations with other companies. Medical Affairs groups work on building awareness... patients, shaping discussions with insurance companies about the pricing and coverage of drugs, and developing... teams, reflecting the need for all parts of a company to collaborate whenever it faces an important challenge...investments over the next 3, 5, or 10 years. Complicating these challenges is the fact that they occur...their jobs most, get pleasure from taking up such complex challenges, quickly breaking them up into smaller...

...derived from the more complex type III-A and III-B systems. Though it was composed of many protein subunits...might be phylogenetically classified as the more complex Class I systems that contain multiple subunits,...evolution, were also working on the project. Using computational observations, Koonin and Makarova found that...the domains into a single protein, shedding the complexity of its ancestors. They classified the system ...system contains two pieces: a single protein, computationally described as a fusion of four Cas7 proteins...significantly reduced amount of off-target toxicity compared to other RNA editing methods — streamlining experimental...mammalian cell lines. However, in those same comparisons, the AbuGoot lab reported no detectable effect...

...Table 1: Taking it a Step Further - Big Company v. Small Company Big (e.g. Pharma) Small (e.g. Biotech...advancement in one company, good performers in for the long haul Limited movement in one company, likely will...academic silo to tenured professor.  Non-academia encompasses millions of choices including pharma, tech transfer...options in nonprofit science. No one list can ever encompass them all. We can’t designate non-academic jobs...and teach students Collaborations may be less “complicated” - for example, easy to initiate a collaboration...More sources of training and resources within the company Must find own sources of knowledge often externally...every 3-6 years—a short and exciting ride Complicated matrix structures are common Small integrated...

...protein. Proteins located in membrane-bound subcompartments, like the mitochondria or nucleus, will likely...loading buffers, as most Bradford assays are not compatible with detergents like SDS, while BCA assays are...their differing chemistries. You therefore cannot compare Bis and Tris gels or blots to each other. Once ...protein-based blocker is either degraded or not completely mixed together.   Commercially available chemical-based...lower cost, and smaller size. However, HRP is not compatible with sodium azide, a common microbial agent used...phosphates have a linear reaction rate and are compatible with antimicrobial agents. Chemiluminescence ...chemiluminescence enzymes, they are more likely to be compatible with stripping and probing.   Imaging At the ...

...easier to identify by comparing peak positions to the base expression. When compared to the control — which... voltage has been adjusted appropriately and compensation performed. Before you read out your plots, you...to make sure you’ve also adjusted voltage and compensated your colors. Here are some good practice tips...population cluster would be shifted to the right in comparison, say somewhere between a 104–105 signal intensity...place your gate based on the dot plot would be complicated.  Figure 2: (A) a smeary dot plot showing...with the baseline suggesting this sample has a comparable expression to the baseline/negative control. ... populations in the histogram plot in Figure 4 compared to the dot plot in Figure 2. Quantification using...

...protein:DNA complexes with antibodies specific to your protein(s) of interest. Once the complex is captured...protein:DNA complexes and finally (5) isolated the DNA from your antibody:protein:DNA complexes. Your DNA... the first step is to ensure your protein:DNA complexes will remain bound to each other until you are ...reversible bonds. This will keep the protein:DNA complexes together until you want to break them. Crosslinking...digestion can preserve more of the protein:DNA complexes. Once you’ve finished fragmenting the DNA, spin...that?  So while this method is certainly long and complex, you may be thinking, well, these are mostly fairly...techniques. What’s all the fuss? Well, ChIP’s complexities come not from the techniques used, but from ...

...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... 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...sense approaches that will prepare you for the competitive landscape beyond college. They will also serve...

...career at a biotech company, I always assumed I’d work one or two years at the company and then go to grad... the head of software product management at my company was open to meeting with me and explaining more...software product manager helps ensure that the company or software group is building the software tools...Well, I’ll give you an example. At my previous company, Ginkgo Bioworks, our software development team...backgrounds, most did not. It was my job to translate complex lab operations workflows into a format the developers... to solve is clear, but the solution is more complicated. You’ll need to be creative to be able to work...framework If you can’t find internships at science companies, try to find internships with translatable skills...

...requirement if you work for a private company you must show that your company employs at least 3 full-time people...Within these sectors, you could start your own company, work in a bench research career or a non-bench... to find the perfect job immediately after you complete your Ph.D. or postdoc. Foreign national researchers... need to be aware of the April 1 deadline for companies to submit an application on their behalf.  If ...technology transfer, communications, medical writing, compliance, core facility management, collaboration management...be directed to you specifically. You must also complete the peer review for it to be counted as proof ...your CV and bio sketch whenever you have new accomplishments to add. Obtain copies and translations of documents...

...challenging for sequences with complex secondary structure. Today, multiple companies offer long single-stranded...ribonucleoprotein (RNP) complexes of Cas9 protein and the separated guide RNA components (crRNA and tracrRNA...months. With CRISPR (B), you can inject CRISPR components directly into a zygote and obtain a knockout ...maximum Easi-CRISPR efficiency. Easi-CRISPR is compatible with standard microinjection and electroporation...successfully used guides targeting the same or complementary strands, as well as guides facing towards or... simple method for delivery of genome editing components to the germline. In contrast to standard microinjection...placed to mediate electroporation. Ohtsuka et al. compared traditional microinjection and i-GONAD knock-in...

...engineered ESCs), and chimeric adults mated to produce completely transgenic offspring. Although powerful, this... genome, the CRISPR/Cas system makes use of 3 components, a guide RNA (gRNA) of about 125 nt that specifies...gRNA, Cas9, and donor oligonucleotide or plasmid components are brought together and microinjected into either...eggs. Or, to avoid handling embryos ex vivo, the components can be electroporated into the oviduct of pregnant... major advantages of CRISPR mouse editing when compared to more conventional methods. First, one can work... work with almost any strains of mice, as compared with conventional gene targeting, which is limited ...including 129 and C57BL/6, for which we have germline competent ESC lines. Second, the process is much quicker...

...ask your interviewers You’re interviewing the company as much as they are interviewing you, so come prepared...performance evaluations, professional development, and company culture.  It’s also a good idea to prepare 1-2 .... This is a chance to highlight your prior accomplishments and demonstrate your science communication ... Connect your prior work or a new idea to the company’s area of expertise. This has potential to make ...consider bringing your own laptop as a backup, if the company will allow it. Before you start your presentation...interview. Expect to be tired once the interview is complete Industry interviews are long! Mine lasted 4.5 ...read emails from HR carefully because sometimes companies have relaxed dress codes for interviews. You can...

...MiSeq that takes about two days to complete. After the run completes, we begin our assembly process. Again...sufficient quantity for sequencing. This process is completed in a plate format yielding anywhere from two to...Illumina sequencing on our newly donated MiSeq.  For Complete Plasmid Sequencing we perform a 2x251 run on our...annotated Genbank files that are useful for more complicated plasmids, like those that contain synthesized...mutations, truncations, and insertions that could compromise function. When we do find mutations, we check..., NGS for some plasmids will not result in one complete, circular assembly. Most of the time, these plasmids...obtain an NGS result that is useful, but not 100% complete, we will still make this data available as a partial...

...your product’s web page, or in the company’s FAQs? A lot of companies and organizations selling biological...answering help emails and phone calls, I feel compelled to share my years of accumulated wisdom so that...information, but it’s not in the best interest of any company to hide information about their products from you...which case, you should probably stop using that company). Frequently asked question pages on website are...customers (and I expect this is the case at most companies). Always go back to the source. Although many...experience: Make sure you’re contacting the correct company. This might seem like a no-brainer, but it happens...please share the data. Maybe not all, but many bio companies have real scientists answering their technical...

...can we compare these methods in specific applications, we may also be able to use these comparisons to enhance...to 9 months in mice [2]). Low Immune Response: Compared to other viral vectors (e.g. Lentivirus, Adenovirus...is possible to use AAV constructs with self-complementing structure (scAAV) (7). As single stranded DNA...mapping of circuits In our lab, we use AAV-DREADD complexes to study neural function. Often the reporter proteins... proteins associated with these complexes are so robustly expressed that anatomical assessments can be...Ready-to-Use Retrograde AAV Preps at Addgene 2) Comparison of genetically engineered receptors (i.e. chemogenetics...neural control. Surprisingly, little research has compared and contrasted optogenetic and chemogenetic receptors...

...reaction or can be purchased 5’-modified from a company. However, unlike an RNA guide, it cannot be produced...probably while it is being synthesized) can form a complex with an ssDNA guide. Therefore, researchers must...inject cells with in vitro preassembled Cas9/gRNA complexes or with guide RNA- and Cas9-expression plasmids...survey and asked researchers if they had tested and compared NgAgo head-to-head with CRISPR-Cas9. The survey...respondents (transfection method, amount of each component per transfection etc.). Take Pooran's NgAgo Survey...same method used by Gao et al, 2016 (1). View the Complete and Up-to-Date Results of Pooran's NgAgo Survey..., leaving a small amount of active NgAgo/ssDNA complex in the cell. As proposed in the original article...

... a Type I-F CRISPR-Cas system. The four major components required for RNA-guided DNA integration are (... (Fig. 1A, top):  A CRISPR RNA (crRNA) comprising a 32 nt spacer sequence specifying the target site....sequences (R and L ends).  Figure 1: (A) Components involved in each type of CRISPR-Transposon system...multiple Cas proteins to form the Cascade targeting complex is a hallmark of Type I CRISPR-Cas systems, in ...nuclease activity. Instead, it forms a novel co-complex with the transposition protein TniQ for targeting... by the system. Guided by the crRNA, QCascade complexes survey the genome and bind target DNA sequences...pairing. Separately, TnsABC forms a transposase complex with the mini-transposon, excises it from the donor...

...library, as well as GC%, reverse compliment sequences, and more. Output_Compact: A text file that describes...there are also benefits gained by lowering the complexity of the screen. For example, the analysis becomes...a lasting impression on me. The slide showed a complex signaling pathway with many receptors, signaling...used these knockout mice to show exactly which components of the known pathway that were essential for ...be very useful for drug discovery, to dissect a complex signaling pathway, and to identify what part of...response to a particular ligand (such as when a component of a microbe or a cytokine is added to a cell)... rapidly performed by targeting all potential components of a signaling pathway. For example, one could...