...to the Cas9/tracrRNA complex. D) The mature crRNA is used to guide the Cas9 complex to the target DNA which...describe the functions of the various CRISPR/Cas9 components. Please note that while this post is intended...intended to provide a general overview of CRISPR components, new Cas9 variants are being discovered all the ... thereof. This individual protein has all the components necessary to: 1. Bind to a guide RNA The guide...remainder of this post, we’ll refer to this RNA complex as the “gRNA”. The gRNA contains both the 20 nucleotide...   Now that you know about all the different components of CRISPR, It's time to start thinking about ...

...are reached, how do science labs celebrate accomplishments and their team's hard work? Do succesful scientists...each other. The Church Lab enjoys each other’s company outside of the lab by sharing meals, going on trips...! How does your lab celebrate each other's accomplishments? Any tips on making time to relax and/or enjoy... celebrating accomplishments in the lab... science labs celebrate accomplishments...

...do I know if I will need compensation? To know if you will need to compensate in your flow panel, you ... to apply a compensation value between the two channels (Figure 1A). Applying compensation means you are...channel and needs to be compensated out of APC-Cy7. After increasing the compensation values for APC against..., you would decrease the compensation value.   Figure 1: Compensation example with single color... be fixed with fairly low compensation values (0-50). Inputting compensation values differs between machines...point. Applying compensation to your experimental samples In order to do compensation on your experimental...before (A) and after (B) compensation between APC and APC-Cy7, with compensation panels below each plot....

... good.  First, why are plasmids incompatible? Plasmid incompatibility occurs when multiple plasmids within...Plasmids with the same replicon are incompatible because they compete for the same replication control machinery...2012). This concept is a little more complex than simply competing for replication control factors and ...high copy plasmids, incompatibility due to partitioning is similar to incompatibility due to having the ...plasmids are incompatible? Scientists have developed a system of bacterial incompatibility groups based...belonging to the same group are incompatible. Currently, there are 27 incompatibility (Inc) groups in the Enterobacteriaceae...whether your plasmids are compatible or not.  Using plasmid incompatibility for therapeutic advantage ...

...tests. All sample comparisons Do you really need to know every pairwise comparison within your population...just perform the relevant comparisons using Dunnett’s test, a test that compares all groups to a single ...we will dive into the nitty gritty details of comparison statistical tests – when to use them, what they...your data follows. Fig. 1: Examples of comparable groups with and without equal levels of variance... discuss more in another blog. T-tests T-tests compare the mean of two groups to determine if they are...one-tailed test also works for exclusively less-than comparisons. Paired and unpaired t-tests Samples that come...occasions, there is an experimental group being compared to a standard value (not a control group). This...

...lower than that of the widely used SpCas9 and comparable to SaCas9. As seen with Cas9 orthologs, Cpf1 ...sophisticated genome-wide analysis. At many of its computationally predicted off-target sites, Cpf1 does not mediate...sites, according to Kim et al. Kim et al. also compared the ratio of total off-target to on-target modification...clinical applications require HDR over NHEJ, so comparing HDR efficiency across CRISPR nucleases and NgAgo... Up On Your Cpf1 Background Read About CRISPR Competitor NgAgo Learn How You Can Use piggyBac Vectors ...

...busy in those early years, the founders knew that company culture and enjoying your job were important parts...each other, dressing in costume, and holding competitions (ranging from cook-offs, to corn toss to trivia...in between). Eleven years later,  Addgene is a company of fifty instead of three, and we are sending hundreds...Though there still isn't time for goofing off, the company culture here at Addgene has remained paramount....Most Addgenies reply with an enthusiastic, “Our company culture! Addgene is the best place to work, we ...work, we genuinely enjoy each other’s ideas and company. The Addgene summer picnic This past weekend was... of University Park at MIT.  I’m sure at some companies, employees wouldn’t be caught dead coming into...

...choice for the job compared to T7 ligase (it was—see Potapov et al., 2018). More complex reactions benefited...tools above to complete a 52-piece assembly of a 40 kb T7 phage genome. They first compared 10-piece linear...design GGA overhang sets, particularly for high-complexity reactions? The researchers over at New England...data-driven approach might allow for an increase in the complexity. In 2018, they published a paper profiling four...2022).     Figure 1: Workflow for high complexity Golden Gate Assemblies. Image courtesy of New... three DAD tools used were key for ultra-high-complexity assembly. While the traditional rules of thumb...data-driven approach was needed to massively expand complexity. The three tools developed, outlined below, allow...

... to head NgAgo has a number of unique features compared to Cas9. These include: More targeting flexibility...sensitivity to off-targets More research is needed to compare CRISPR and NgAgo, but it seems that NgAgo has lower...

...lab. How to make your own competent cells It’s easy to make your own competent cells. If you need a high...from purchasing competent cells. However, for most everyday cloning, homemade competent cells will work...OpenWetWare Preparing chemically competent cells (protocol)  Preparing electrocompetent cells (protocol)  Untergasser...Preparation of chemically competent cells (protocol) Preparation of electrocompetent cells (protocol) At...produces 20mL of competent cells, enough for 800 transformations when using 25uL competent cells per reaction...Transformation of chemically competent cells (protocol)  Transformation of electrocompetent cells (protocol)  ...well in other competent cells) to ensure that your cells can be readily transformed. Compare the number ...

...approach to monitor the composition of spatiotemporally defined protein complexes." Nature communications...neighboring proteins. Thus to capture protein complexes, scientists can simply express a bait protein ...domain to allow for promiscuous biotinylation. Compared to BioID, BioID2 exhibits enhanced proximal labelling...mutants, the 35 kD TurboID, which has 15 mutations compared to BirA and the 28kD miniTurbo, which has the ...the N-terminus domain deleted and 13 mutations compared to wild type BirA. TurboID and miniTurbo both enable...interactions Split-BioID combines a protein fragment complementation (PCA) technique with proximity labelling to...vivo technique where two proteins are fused to complementary reporter proteins that can only assemble when...

... a startup company. However, like many like-minded people, I quickly realize that a company requires more... and tested in the “market” by competing in business plan competitions. After much learning, the focus...plan competition and advance investors to the next stage of diligence." In business competitions everything... goals and vision for the company; this will help avoid future complications and potential conflicts that...during graduate school and turn it into a biotech company. Transitioning an idea from academia to a startup...clearly communicate their idea at various levels of complexity and abstraction and convince people with diverse...concept development and participating in business competitions. When the team was unable to secure enough money...

...VPR fuses a tripartite complex with dCas9 to activate transcription. This complex consists of the VP64 ...sequences, CRISPR/Cas systems rely on a guide RNA complementary to the sequence of interest. Upon binding, CRISPRa...rely on fusion proteins while others re-engineer components of Cas systems themselves. SunTag, SAM, and VPR...consistently shows the highest levels of gene activation compared to other CRISPR activators, making it a popular...once), however, SAM exhibits activation levels comparable to other popular activation methods (VPR and ...initial dCas9-VP64 activator, but lower levels compared to the SAM system. Its activation levels are similar...to that of SunTag. An advantage to this method compared to other notable CRISPR activators is that it ...

...Bimolecular Fluorescence Complementation (BiFC). What is bimolecular fluorescence complementation? Bimolecular fluorescence...often easier said than done. Fortunately, a complementary method exists for visualizing protein-protein... fluorescence complementation is a method for detecting protein-protein interactions that uses split fluorescent...fragments that individually lack fluorescence. When complementary FP fragments are fused to two hypothesized protein...interaction partners (PIPs), the FP fragments form the complete FP because the PIPs they’re tethered to bring ...into close proximity (Figure 1). Formation of the complete FP results in a fluorescent signal that can be...  Applications for bimolecular fluorescence complementation BiFC has been used to directly visualize PPIs...

...known PID formats are resolvable compact identifiers, which are Compact Uniform Resource Identifiers (CURIEs... authors comply with journal policies, Addgene now provides an RRID and a resolvable compact identifier...lost (e.g. a supplier is bought out by another company and catalog number changes) the original identifier...the specific plasmids used. RRIDs and resolvable compact identifiers now exist for all Addgene plasmids!...which has the Addgene ID #87155. The resolvable compact identifier format for lenti-Cas9-VQR is the URL...to include the option of citing with resolvable compact identifier and RRID formats. Let’s go back to our...the entire line. Cite plasmid using a resolvable compact identifier and RRID “lenti-Cas9-VQR-Blast was a...

...biofilm formation. The quest for plant compounds to treat complications of diabetes Gloria Otito Izu, PhD...Her goal is to identify and isolate pure plant compounds with antidiabetic properties that can ameliorate...nephropathy, one of the major pathophysiological complications of diabetes mellitus. Recombinant human proteins...expression systems here) and the activity of the plant compounds on these proteins will be tested. Investigating...

...easily design, generate, and test constructs composed of complex genetic elements. To demonstrate the usefulness...several GMAP-compatible vectors for a wide variety of biological applications. First, GMAP-compatible backbones...mammalian systems. The DULIP system is comprised of Gateway-compatible plasmids in which “bait” proteins are...plasmids designed to complement unused auxotrophies in Saccharomyces strains by compensating for histidine (... do so have been hindered by a lack of tools, compounds, and assays to monitor activation of these receptors...including a stop codon) formats. The collection is compatible with both standard Gateway cloning and the FNLCR...assembly platform for generating both simple and complex DNA constructs Have you ever wished you could ...

...replication components are called helper plasmids. Below, we’ll breakdown the vector components first by ...nucleocapsid viral components needed for viral packaging, assembly, and display. Pol, the last component, contains...there are bonus components to these vectors that aren’t reflected in the common components outlined above...lentiviral guide outlines these components.     Fig. 1 - Viral vector components of retrovirus  Adeno-associated...production reasons, multiple plasmids with many components are used to package lab grown viruses. It can... protocols! In reality, there are many shared components across viruses (AAV, lentivirus, etc.) and their... what gag and pol stand for, we demystify all components of viral vectors here! Need Virus? Check out...

...role at a commercial virology testing company. This particular company carries out clinical trials of drugs...treatments for external companies. It was really exciting to work in a larger commercial company; the pay was good...the company and that my ideas and changes were welcomed, which is a benefit over larger companies which...obtaining work in scientific research would be very competitive, and so I applied for a summer role as a Research...This was particularly interesting as I had just completed my thesis on the study of e2Fb, which is a tumour...without issues. Because the primary purpose of the company was to fulfill contracts, my work sometimes felt...strong point. Working in industry: virology On completing my MSc, I again had to look at what type of work...

...different components, but all contain a FRET pair, a genetic component, and a small molecule component. Early...developed voltage sensor with a genetic component. The VSD components have historically come from a variety...have been engineered to have kinetics comparable to its one component counterpart and is significantly brighter...cellular localization component is typically handled by either the fluorescent component (i.e., when the dye...tools have two main elements: a voltage-sensing component and a fluorescent output. The GEVI family of gadgets... a hybrid approach and utilize both a genetic component as well as a small molecule which we will describe...reporter and the mechanism of their voltage sensing component, which separates them into two distinct categories...