...proteins. lon protease: Strains where this is completely deleted (designated lon or Δlon) similary reduce... and a p15A origin of replication, making it incompatible with other p15A plasmids. pLys comes in two ...measures that can help get you back on track: Compatibility: Double-check your plasmid backbone and expression...expression strain to make sure they are compatible. An arabinose-inducible plasmid will not express in an...strain for example, nor will a p15 plasmid be compatible with a pLys strain. Your strain may require additional...better at 30°C or room-temperature, which is accomplished by growing your culture to the desired density...

...metabolites. Biotin acts as a carboxyl carrier from one compound to the other. These biotin dependent enzymes are...discovered in the 1960s, strepavidin. The Biotin-avidin complex can then be used and/or manipulated for a scientist...immunoprecipitation experiments. Biotinylated proteins or complexes can be easily purified using columns or beads ... sample. Any free biotin-binding sites on this complex can bind to your biotinylated antibody. This creates...creates a large meshwork of biotin-avidin complexes bound to your protein of interest leading to amplification...This strategy is referred to as the Avidin-Biotin Complex (ABC) immunohistochemistry (IHC). For more information...

...across the entire blot. So how can you accurately compare different lanes? In relative quantification, the... conclusions, and it’s also problematic for computational quantification. Outside the linear detection...individual protein. This ensures you are truly comparing the abundance of your POI as a fraction of the...detection ranges of multiple targets. Convenient! Comparison of loading control approaches Cartoons are nice...all, but we’ll leave you with this real-world comparison of different loading control approaches from ...different methods of quantifying total protein are compared. While a higher slope indicates higher sensitivity...

... different universities are trying to compile more comprehensive demographic information about the postdoc...Postdoc: An Institutional Perspective. This lack of complete information does not, however, prevent us from...from the career panel was that there are many companies and organizations (including Addgene), that need...scientifically minded, and able to communicate complex information - all traits you are likely to find...others within their labs - as a sort of over-compensation, these mentors can treat the underrepresented...

... received his PhD in Computer Science from MIT. His research focused on computer systems and networks.... 4 - Developing Addgene as a modern e-commerce company and establishing credibility Chapter 5 - Realizing...Kenneth received his Bachelor of Science degree in Computer Engineering from Tufts University and his Master...and a Masters in Engineering from MIT, both in Computer Science.   Special thanks to our sound engineer...

...bacterial CRISPR engineering One common way of accomplishing bacterial genome engineering is done with recombineering...transposition. INTEGRATE consists of four major components including (1) a CRISPR RNA, (2) four proteins...transcription by physically blocking the elongation complex from binding the DNA or extending the transcript...transcription factors, phage proteins, and RNAP components (Dong et al., 2018). They found a transcription...transcription factor called SoxS for CRISPRa. Adding complexity, they found that they could also include a CRISPRi...systems is base editing. This process is generally accomplished by fusing an enzyme capable of converting one... targeting approach is then used to direct the complex to a specific locus. This approach provides a more...

...Drs. Koeris and Lu founded the biotech startup company, Novophage. As you'll hear Dr. Koeris explain however...Ventures, Koeris helped change Novophage's focus. The company went from developing therapeutics to developing...pathogens in food products. Along the way, the company changed its name from Novophage to Sample 6. To...using Sample 6 and why? 24:41 - 29:00 : Dr. Koeris compares and contrasts being a graduate student with being...personally take on the risk of founding a startup company? 32:20 - 34:37 : Whose responsibility is it to ...

...reporters Difficult to Multiplex: FRET reporters are comprised of two fluorescent proteins with very specific...encoded fluorescent kinase reporters. A KTR is comprised of a fluorescently-tagged substrate for a kinase...for kinase activity.  Advantages of KTRs When compared to FRET reporters for measuring kinase activity...of KTRs KTRs also have some disadvantages when compared to FRET reporters. First, KTRs monitor global ...measure kinase activity in specific subcellular compartments. Second, because KTRs shuttle between the nucleus...

...possibilities in the engineering of complex gene networks. A comparable system for targeting specific sequences... of RNA is highly desirable for extending the complexity of genetic circuits, allowing for tighter spatio-temporal... protein that binds an alternate sequence. By comparison, Abil explained, the specificity of other RNA..., designer PUF proteins can be fused to other components to lend them many other functions for use in ...

...markers. These are especially useful in protein complex studies where tagging of multiple protein products...for easy capture and detection of proteins and complexes, without the artifacts sometimes associated with...Formosa, at the University of Utah, has built a complete collection of yeast tagging modules with each ...ideal for tandem affinity purification of protein complexes. John Pringle and Jürg Bähler have deposited a...GST, or GFP). Longtine et al.(3) describe a complimentary set of plasmids for use in S. cerevisiae, with...

...The application process When I first saw the comprehensive job description for this co-op, I was immediately...description of the tasks I would be responsible for, in comparison to the other positions listed with a much less...training, I was gradually introduced to Addgene as a company and given more details on what my position entailed...they do and how it relates to other teams at the company. This provides new (and old) employees with information...Northwestern University. She is one of several students completing their co-op at Addgene. This is her second contribution...

...without the exo/polymerase generation of annealed complementary regions. Why use a high fidelity ligase? Numerous...the Ligase Detection Reaction (LDR), probes complementary to the sequence of interest are annealed to ...separate pools were prepared, each with a different complement strand covering all 16 possible NN pairs in the... system for direct identification of allelic composition of mutated DNA in a mixed population of excess...JS, et al. "A high-throughput assay for the comprehensive profiling of DNA ligase fidelity." Nucleic acids.... Pascal, John M., et al. "Human DNA ligase I completely encircles and partially unwinds nicked DNA." ...

...Visualize components of individual cells Separation/identification of fluorescently-labeled components Fluorescence-activated...process or detect a given molecule. The sensor component is usually a protein that undergoes a conformational...biosensors can be targeted to a particular cellular compartment such as the nucleus, mitochondria, endoplasmic... biosensor: which fluorescent protein is most compatible with your lab and experimental setup? Consider...

...Palindromic Repeats. That contrast of simplicity and complexity is reflected in the biology, too. CRISPR is an...proteins, and a CRISPR array. The CRISPR array is composed of identical repeat sequences and variable spacer..., and the spacer sequence is used to target complementary DNA sequences. In laboratory settings, an sgRNA...whose sequence is not checked. Assuming full complementarity, its sequence matches the guide sequence. PAM...either crRNA or sgRNA). Cas9 requires perfect complementarity in this region between the guide and target...

...give you the reverse sequence, complement sequence, or reverse and complement sequence.  Filter DNA tool:...well as those deposited with Addgene. NCBI BLAST: Compares nucleotide or protein sequences to sequences in... tools help with that.  DNA sequence reverse complement tool: Enter a DNA sequence and the program will...DAD): This tool designs overhang sets for high complexity Golden Gate Reactions Cloning ligation reaction...

...response to a viral infection a) antibody-dependent complement deposition (ADCD) b) antibody-dependent cellular... Erp, et al., 2019.    Antibody-dependent complement deposition (ADCD) Antibodies can bind to a pathogen... known as opsonization. In antibody-dependent complement deposition (ADCD), opsonization of IgG and IgM...pathogen activates the immune system's classical complement pathway, starting a cascade of events that results.... PMID: 10742152. Dunkelberger, J., Song, WC. Complement and its role in innate and adaptive immune responses...

...Development Manager at eScience Labs, an educational company that creates hands-on science lab kits and digital...from.” Working at eScience Labs, an educational company that creates hands-on science lab kits and digital... editing.” It was for Cactus Communications, a company that provides scientific editing services. “I was... stumbled upon eScience by luck, by chance, by complete happenstance. It was on Indeed or one of the job...what it’s like to work for a science education company. And make sure to subscribe to the Career Channel...

...hand. Records of plasmid construction were often incomplete or nonexistent. In the 21st century, many molecular...molecular biologists didn’t know the complete sequences or properties of the DNA molecules they were using...has been thoroughly answered by experts in human-computer interaction (HCI), and we have adhered rigorously...what the user wants to do and make the path to accomplishing their goals as intuitive and painless as possible...who uses SnapGene or the free SnapGene Viewer companion product. If you don’t use SnapGene yet, you can...

...when Neil Thompson, a research scientist at MIT’s Computer Science and Artificial Intelligence Lab and a ...properties (GC content or rare codon percentage) and comparative properties (percent sequence identity and percent...query coverage) that could be determined through comparisons with a reference sequence database. Through a...content between many organisms and DNA synthesis companies often work within a narrow range for GC content...synthetic vs. natural gene are essential and complementary tools in a time where biological engineering...

...30678704. Sparse cell labeling using recombinase competition Article contributed by Aliyah Weinstein Sparse...Sparse Predictive Activity through Recombinase Competition (SPARC) is a fully genetic tool for labeling just...heat-shocking the organism. The SPARC system uses competitive recombination sites to selectively remove a stop...microscopy, they also show that this method is compatible with dead cell staining using propidium iodine...purification of tTALE-eGFP and tTALE-mCherry. In comparison to intercalating fluorescent dyes which can cause...

...build and grow the gene therapy division of the company.  Ultragenyx’s goal is to develop novel products...manufacturing and quality, especially with regard to compliance with health authority regulations,” Baradaran...Belgium. “That’s where I not only got incredibly compelled by wet bench molecular research, but also became...grants, or setting up commercial and nonprofit companies. His lab focuses on gene therapy work for genetic...these inefficiencies is pretty remarkable. I was compelled to be part of that.” He’s deposited AAV constructs...

...between two opposing forces or (2) the result of competing desires or (3) the presence of obstacles that ...until you don't, of trying to understand your completely unexpected findings, or understanding why your...thrice-failed experiment, this paper was actually from a completely unexpected finding in a different experiment,...your conflict, you’ll need two other things to complete your story: an introduction, and a resolution....conflict to get your audience invested in the more complex story, and then build on that in your resolution...

...sufficient to give specificity and to also be compatible with a standard PCR reaction (see PCR Video)....design is similar, but we need to use the reverse complement to get PCR amplification. We can start similarly...the ORF). We now need to generate the reverse-complement of this sequence so that we can successfully ...amplify the ORF. You can generate the reverse-complement using existing software (a quick internet search... out Our Plasmid Reference Pages Deposit Your Completed Plasmids Find the Perfect Plasmid for Your Research...

...Zalatan JG, et al.'s recent paper, "Engineering Complex Synthetic Transcriptional Programs with CRISPR ... the eukaryotic cell for the execution of many complex cellular behaviors, such as differentiation or ...customizable single guide RNA (sgRNA) that bears complementarity to a 20bp target sequence (Qi et al. 2013)....appealing since the RNA scaffold modules are comparatively small allowing multiple sgRNA constructs to .... (2013). PubMed. Zalatan, J. G. Engineering Complex Synthetic Transcriptional Programs with CRISPR ...

...downregulate the expression of the major histocompatibility complex (MHC) class I proteins on their surfaces...immune response to the development of tumors is a complicated and dynamic phenomenon that is interestingly ...rear its head once the body’s immune system is compromised either due to age, infections, or other strenuous...focus on include: Cell heterogeneity Tumors are composed of a wide variety of cell types. This cell heterogeneity...successful tumor progression. A tumor’s genetic composition is constantly in flux and is dependent on the...

...1); similar to an MRI or CT scanner. This is accomplished by focusing excitation light to a point in the... bleaching) to a single focal point. This is accomplished by using, for example, red light instead of ... photons of red light are needed to excite GFP compared to only one of blue. Only at the focal point of...coverslip and a sample in aqueous buffer it is completely reflected. This reflection occurs due to the ...immersed in. Although the excitation light is completely reflected, energy is propagated into the sample...cover samples of this size, those that can will completely image large tissues in a few hours. Although ...

...and some home-made equipment. It was humble in comparison to the labs you’d find in Kendall square, but...Most of our equipment has been donated by local companies and BosLab has grown into an organization consisting... Timothy Stiles joined BosLab last year with a computer science degree and had zero pipetting experience...yeast to glow, produce synthetic scents, detect compounds in the environment, and openly publish protocols...the local community (including local garden and compost clubs) to participate in this study. How to get...

... other hand absorb best at 280 nm and organic compounds and chaotropic salts maximally absorb at 230 nm...SYBRGreen, are specific for double strand DNA (as compared to spectrophotometric methods that measure all... corresponding fluorescence. That way, you can compare the fluorescence of your sample against this curve...relative to the intensity in the ladder. It’s best to compare the band intensity of the fragment in the ladder...sugars under acidic conditions and forms a blue complex that can be quantified at 595 nm. This method is...

... precision gene editing. CRISPR-Cas9, the key component to enable efficient base editing and prime editing...unable to process its own array but is the key component to enable efficient base editing and prime editing... or any other kinds of CRISPR tools, without compromising their strategic advantages in gene editing, ...higher Cas9-independent off-target editing in comparison with single-site editing. Encouraged by the results...#186715) and hCtRNA_VT (#186716).   Fig. 2: Comparison of previous representative dual-gRNA architectures...

...equivalent of crossing off days on the calendar after completing your writing goal, which can serve as positive...graph of which days of the week you most often completed your habit is useful for figuring out when to ...makes suggestions on how to make your writing less complex. While I don’t think you should accept all of the...jargon in your writing. While it’s not possible to completely avoid jargon, this tool can help you avoid overusing...collaborators. Zotero is a desktop based program that’s compatible with Mac, Windows, and Linux.  Mendeley and Zotero...

...commonly used yeast by biotech and pharmaceutical companies is K. phaffii (formerly known as pichia). What...general research community. Phillips Petroleum Company first commercially developed K. phaffii in the ...Since then, multiple biotech and pharmaceutical companies (along with some research laboratories) have picked...of the uses outlined below. Biology They say comparison is the thief of joy, but I think it’s acceptable...10.3389/fmicb.2020.607028  Riley, R., et al., Comparative genomics of biotechnologically important yeasts...

... potential binding affinity to major histocompatibility complexes (MHC class I and II).  There are multiple...of adverse reactions In silico assessment uses computer modeling and predictive algorithms to forecast...approach The in silico approach cannot replicate the complexity of antigen processing events that take place ...in-vitro models can be built that contain various components of the immune system. Figure 2: In-vitro...

...need to be decided. Figure out what you need to accomplish to get to the next stage, and then adjourn. ... if any additional resources are needed. All-Company Meetings: As your organization grows, so will the...to address one point aloud. This keeps our all-company meetings short, informative, and pleasant. Email... positive peer pressure to make sure everyone completes their assigned items to keep the project moving...timely feedback on each other’s projects. For all-company meetings, monthly still works best. Figure out ...

...Cas9.  For a brief comparison of Cpf1 vs. Cas9, see the table below. Table 1. Comparing the Cas9 and Cpf1...simplified system for editing multiple targets compared to Cas9.  Read on to learn more about Cpf1 multiplexing...Cpf1 CRISPR Nucleases   Cas9 Cpf1 Components crRNAtracrRNACas9 crRNACpf1 Endonuclease size ...expression vector.  See the graph in figure 2 for a comparison of editing frequency resulting from transfection...

...transcription to begin. In addition to arabinose, cAMP complexed with cAMP activator protein (CAP) can also stimulate...another way to activate gene expression, and two-component systems used in synthetic biology use light to...Addgene also has yellow flame light-regulated two component systems designed by the Tabor lab. Which inducible...crossover. The induction mechanism must also be compatible with your experimental system. Leakiness If you...Region Plasmids 101: Cre-lox Light-Switchable Two-Component Systems for E. coli Browse other blog posts from...

...Anti-FLAG frankenbody (FB) construction from the complementarity determining region of an anti-FLAG scFv and... variations in DR sequences. Figure 2: comparison of Cas 9 vs. Cas 12a expression cassettes when... of performing similarly to Cas9 screens in a compacted library. Overall, Cas12a’s ability to target multiple...CRISPR-Cas system for editing and gene activation, is composed of only 529 amino acids, less than half the size...or Cas12a. Despite its small size, CasMINI is comparable in efficacy to dCas12a systems in both gene activation...

...Cas enzymes and variants available. Without a comprehensive resource, it can be overwhelming to choose a...classification system, CasID, to help facilitate direct comparison of different enzymes. CasPEDIA is meant to be...how can I multiplex this enzyme? What other RNA components are required? CasPEDIA provides information on...gRNA design, etc.). With this feature, you can compare different Cas enzymes to make sure you are getting...isn’t always the winner!).   Fig. 3 – Comparison of different Cas enzymes using CasPEDIA. Adapted...

... You can do more than just ‘sense’ biological compounds and reactions; you can quantify them with biosensors... how to use viruses to set up these systems. Components of a biosensor Some of the biosensors used in... that you are interested in. The most obvious component of a biosensor is the thing that you care about...or an electrolyte such as calcium. This varied component, which we will refer to as the analyte, needs ... fluorescence, pH change, heat, etc. The last component of the biosensor is the signal transducer which...

...researchers new to GCE aren’t able to successfully complete their experiments and many others are dubious ...’t give it a try. Optimization Why is GCE so complicated? Well, while the techniques are straightforward...tools are not very robust. When using GCE, each component of the experiment system (including media, cell...optimization parameters the same for all three components, meaning that the final protocol may involve ...has “10x the impact of PCR. But it’s 100x more complicated.” And Ryan Mehl would know. He’s the director...

...be incorporated into these constructs without compromising cleavage efficiency. In addition, tRNAs contain...generation for two PTG constructs in rice protoplasts. Compared to matching traditional gRNA-expressing constructs...examined editing efficiency in intact rice plants, comparing traditional gRNA constructs with PTGs. In PTG ...frequency at both targets (35% and 76% biallelic), compared with only 44% and 66% indel frequency with the...in the graphic below - including some simple comparisons. The easy multiplexing afforded by PTGs will...