...reactions. The BP reaction creates an attL-flanked entry clone. The LR reaction creates an expression clone ...reactions. Recombination of attP and attB sites creates attL and attR sites.   Gateway vectors contain...Gateway plasmids.    Figure 3: Method A to create an entry clone: recombination of an attB-flanked...attL-containing entry vector.   Figure 4: Method B to create an entry clone: TOPO cloning the insert into an...attL-containing entry vector.   Figure 5: Method C to create an entry clone: Restriction cloning the inset into...vector will recombine with the attL-entry clone to create the expression clone.    Figure 6: Generating...between the entry clone and destination vector creates two products: the desires expression clone and ...

...vesicles with nonlinear contrast. Increasing expression and decreasing genetic footprint We took a cue ...heterologously by bacteria inside the body. So we screened gas vesicles gene clusters from a diverse set ...expression heterogeneity required single cell screening to isolate a clonal cell line capable of producing... expression to appreciable levels. We wanted to create second-generation mARGs with higher expression,...reduced the number of copies of gvpA needed to increase expression. We were able to express Anabaena ...expression leads to greater amount of gas vesicles and increased ultrasound contrast. In all the cell lines we ...their tracking with ultrasound imaging (Fig. 2d). Creating a cell line can be a time-intensive endeavor. ...

... we grew. As the number of deposited plasmids increased, the accompanying sequencing data collection and...bioinformatics workflow management system As novices to creating a robust data pipeline that would be reliable ...community of Nextflow supporters existed who had created a shared resource of pipelines for genomic data... with others without any complicated setup. We created, tested, and debugged each of our pipeline steps...running the pipeline locally and in the cloud. Creating a bioinformatics workflow in Nextflow Establishing...scripting language that can be executed on Linux, we created our first process by simply copying and pasting...was working as expected, we moved forward with creating the next process and linking the output from the...

...polymerase, 2) increased PCR extension time, 3) increased concentration of MgCl2, 4) increased concentration...PCR reaction is created by making some or all of the following modifications: 1) increased concentration...saturation mutagenesis Several methods can be used to create mutational scanning libraries. While each of these...Therefore, Gibson assembly is better suited for creating small, focused scanning libraries. PFunkel:...library that’s ready to be tested in a functional screen of your liking. This method is similar to site-directed... screen...

...greatly reduced costs per plasmid. We generally create kits to allow you to easily use a set of plasmids...markers built into all plasmids in the kit help decrease false positives observed in the cloning process...protein combinatorial library by in vivo functional screen." Scientific reports 5 (2015). BIOFAB plasmid ...set 3000 Series Confronted with the problem of creating vectors for predictable protein expression in ...bacteria, researchers at the BIOFAB and the Endy Lab created this kit. The plasmids within it contain a variety...assembly. PhD student Dejan Gagoski used SITS to create the pCell-Free vectors, Gateway-compatible backbones...these different modular repeat segments, you can create a PUF protein that binds your RNA sequence of interest...

...was identified from a high-throughput screen, selected for increased voltage response, brightness, and photostability... They use a unique and powerful high-throughput screen to select variants based on performance metrics...resulting JEDI-2P has faster response kinetics and increased brightness, sensitivity, and photostability under... be generalized to other RTKs of interest, they create sensors for FGFR1, EGFR and ErbB2. And orthogonal...developed using a high-throughput multiparameter screen (Koveal et al. 2022). Lactate is a key metabolite...cellular concentrations of lactate. The microfluidic screening platform developed here is also a promising approach...Figure 6: A) Schematic of BeadScan platform for screening candidate biosensor variants for performance under...

...as a stepwise decrease in signal strength in a flow plot. d = days post treatment. Created using biorender.com...binding and (b) antibody host species interactions. Created using biorender.com. Another way of indirect...1b). As above, the advantage of this system is increased flexibility for panel design. Another perk is ...can be bound by several secondary antibodies, increasing the fluorescence signal intensity. However, you...cell, CD4+ T cell, dendritic cell, CD8+ T cell. Created using biorender.com.   Intracellular labeling...cytosolic proteins, as well as proteins of the secretory pathway. Those can include cytokines and chemokines...bind to their targets in the cytosol and nucleus. Created using biorender.com.   Dye stains Dye stains...

...topics discussed during the symposium will prove incredibly important to the stability of the biological ...align trainee expectations with job outcomes, and increase diversity in the academic research environment...these problems and some that stuck out to me were: Decrease the Number of Postdocs De-incentivize going into... more services based on what they're spending. Increase the number of science masters programs to prepare...postdoc salaries as opposed to Staff Scientists Increase the Starting Salary for Postdocs to $50,000 (the...salary as reported by Paula Stephan is $40,000) Increase Knowledge about Postdoc and Graduate Student Career...was highlighted during the conference that just increasing funding for academic research will not solve ...

...payload to a cell but cannot create new particles. As a bonus, this increases the carrying capacity of the...non-enveloped virus (left) and an enveloped virus (right). Created using biorender.com   If you’re new to talking ... Figure 2: Baltimore replication classes. Image credit: Louten, 2016 Viruses with DNA genomes can either...After a virion infects a cell, the single particle creates many copies of itself, sometimes up to millions...the risk of infection — researchers were able to create far safer viral vectors that can be used in BSL... the viral vector, increasing both safety and usefulness of the tool. In fact, most gutted genomes have...have as much genome removed as possible, just to increase the viral vector’s carrying capacity. In order...

..., the Liu lab created and evaluated 19 PE1 variants with RT mutations known to increase activity, enhance... initial nick site (to avoid creating a double strand break), increasing editing efficiencies 2-3-fold... Figure 1: An overview of prime editing. Created with BioRender.com. How does it work? First... between the template and primer binding site, increase processivity, or improve thermostability. What...fold. However, the process of double nicking increases indel formation slightly. Designing the sgRNA with...inventors kept optimizing it. Using a pooled CRISPRi screen, the Liu lab discovered that cellular mismatch ...addresses the problem of RNA stability. Using a CRISPRi screen, they identified a protein that enhances prime ...

...site-saturation mutagenesis, and medium-throughput screening, she increased the affinity of the sensor for serotonin...overwhelming response and were excited to see the many creative uses of fluorescent proteins. We’re happy to announce...thousands of protein variants simultaneously  With increasing sequencing power, scientists are able to quickly... protein-coding pathogenic human variants are decreased in protein abundance, possibly due to misfolding...neuromodulator, and evidence suggests that it is incredibly important for nearly every aspect of life.” Unger...system. “This bottom-up approach will allow me to create a fully controllable and reproducible set up,” ...explains. Developing an artificial leaf replica means creating a “leaf” with similar permeability of water and...

...physically joining multiple fragments of DNA to create a synthetically designed DNA sequence. There are...fragment joining, but this is fine for its goal of creating a choice of standardized constructs and tools ... regions. Golden Gate Assembly also allows the creation of standardized DNA constructs, but its use of... expediently join multiple synthetic fragments, create multiple mutations in one or multiple fragments...well-designed fragment overlap sections, while still creating scarless plasmid inserts.  Want to Learn More...and eliminates the annealing step usually used to create a double-stranded oligo. Generating an sgRNA construct...directly transformed into E.coli for plating, screening and generation of sgRNA. Generation of sgRNA libraries...

...lower its aggregation tendency, increase its brightness, and decrease the amount of light required for...Fluorescent proteins have enabled scientists to pursue creative research avenues previously unavailable to them...applicants but Xin and Fatima stood out for their creativity in the use of fluorescent proteins and the potential...Fluorescent proteins can be applied in a long list of creative ways. In her graduate work in Michael Lin’s lab...by using photoswitchable fluorescent proteins to create enzymes that can be turned on and off with light...researchers will be able to use similar designs to create a wide variety of enzymes and proteins with inducible... to produce HMOs, sensor binding results in an increase in fluorescence of cells that actively produce...

... primary screen to knowing what are the real hits is so much shorter…. it’s a great screening technology...come here, I need to spend time here, this is an incredible place.” Doench has spent most of his research...researchers in a variety of fields develop RNAi screens to answer their own research questions. Starting... For his post-doc, Doench worked on using RNAi screens to learn more about cancer biology in Ed Harlow...developing CRISPR pooled libraries for use in genetic screens and has contributed much of his knowledge to the...was completely justified… it has been just an incredibly useful technology.” He goes on to explain that...become more efficient in terms of editing and screening) such that thousands of individual edits can be...

...this approach can greatly increase the sensitivity of an assay by increasing the number of signaling molecules...green; and proteins are represented in orange. Created with BioRender.com.    How do secondary antibodies...shorter than that of the primary antibody, due to increased binding affinity, many indirect antibody protocols...are designed to take place over multiple days. Increased run time is considered one of the major drawbacks...specific isotype subclasses of primary antibodies can increase the possible number of target proteins.   The ...immunohistochemistry advances have found ways to increase signals from conjugated primary antibodies. The...as nanobodies, has introduced new options for increasing signal strength.  For the time being, though,...

... the untagged allele. Created with BioRender.com. The first step in creating the plasmids was to identify...be prohibitively expensive for many labs. We’ve created and tested plasmids that use CRISPR/Cas9 to endogenously...first exon of the gene. In this manner, we have created an initial set of 10 plasmids representing 10 human...linker, and the GFP tag sequence to be inserted. Created with BioRender.com. A key feature of our methodology...the double strand break and introducing the tag. Created with BioRender.com. Plasmids, plasmids everywhere...strategy and design described above can be used to create similar donor plasmids for introducing tags into...

... older plates could increase plasmid loss or mutation. Let it incubate Increasing the incubation time ...prep We'll discuss the following techniques to increase plasmid yield. Keep in mind that you might need...particularly stubborn prep. Check the bacterial strain Increase the volume Improve the culture media Let it incubate...more extensive listing of common E. coli strains. Increase the volume Starting with a larger culture volume...you are working with a low copy plasmid. When increasing the culture volume, be aware that a higher volume...origins of replication (e.g. p15A, ColE1) can be increased by adding chloramphenicol to the culture medium...glycerol or glucose (in moderation) may also serve to increase cell density and plasmid yield. Start with a single...

...Laws Bring Increased Postdoc Wages Our most popular blog post this month shares incredibly important information...enacted labor laws will likely increase postdoc earnings in the U.S. This increase, while overdue in my opinion...considering a postdoc. In “Changing Labor Laws Bring Increased Postdoc Wages,” Future of Research executive director...variety of downstream effects including possibly decreasing the number of postdocs various academic institutions...Science Communication Changing Labor Laws Bring Increased Postdoc Wages I hope you all have a great November...

...experiment. Here, we will provide an overview of how to create and use a standard curve and provide some general... I make the standards for a standard curve? You create a standard curve by serially diluting a known control...mix the samples well by vortexing or inversion. Created with BioRender.com.   When choosing a standard... vortexing between each step of the series. To increase the accuracy of the curve, avoid pipetting small...value. How do I plot and use a standard curve? To create a standard curve, you graph the measured response...wider span of concentrations but suffers from decreased specificity, especially as it approaches the minimum... all of the samples in the assay, as this will increase the accuracy of the data. For assays involving...

...enable the creation of CAR-T cells directly within the body. Overall, DIRECTED can increase on-target ...reprogram delivery vehicles like lentiviral vectors, CreVLPs, and eVLPs for in vivo and in vitro applications.... The Ting lab recently made two deposits that increase the utility of this technique.  By engineering... O’Neill The Cohen lab and colleagues recently created a system for recording the transcriptional history...resembling a “ticker tape” recording. The authors created externally-timed bands of color with HaloTag-iPAK4...thin sections of tissue, the Wu Lab is working to create a three-dimensional atlas of protein expression...-95 [K28/43R] (70 µm mounted sagittal section). Credit: Zhuhao Wu, Weill Cornell Medicine.  Interested...