... in yeast Base editing CRISPRi in Caulobacter crescentus Multiplexed gRNA for human pluripotent stem cells...editing. Using several rapid and cost-effective screening methods, David Liu’s lab identified and validated... base editor plasmids CRISPRi in Caulobacter crescentus Figure 1: Serial dilutions of CRISPRi in...adapted from Guzzo et al., 2020. Caulobacter crescentus is a popular model organism for studying the ...ctrA sgRNA and saw that the levels of ctrA mRNA decreased after 20 minutes in xylose (which controls expression...methylcytosine oxidation to DNA repair, which increases activation of methylation-silenced genes. Read...

...experiments but we can definitely give you credit for it and increase your impact! Download ReFigure extension...along with the article metadata, legend and user created notes on a “Pinterest for scientists”? ReFigure...Linkedin and ResearchGate profile. To see how to create ReFigures in a few minutes, visit our YouTube [email protected] Please support ReFigure by creating ReFigures and answering this 2 minute survey! ...impact of teaching and doing science is rapid, concrete and open to innovation by all.  Additional Resources...

... biosensor creation, and custom FRET biosensors can be created using the cpFRET kit created by Olivier...Robert Campbell’s lab display calcium-dependent increases in fluorescence of up to 11,000-fold. FRET biosensors...biosensors. Blue flame “cameleon” calcium sensors created by Roger Tsien’s lab contain two fluorescent proteins...calcium binds, it triggers CaM/M13 interaction that increases FRET transfer between the fluorescent proteins...

...are under a Creative Commons 3.0 license, which requires users to give appropriate credit, provide a link...about new scientific techniques and cool plasmids. Creating graphics were usually the last thing I thought..., I found some free design software that let me create graphics that better communicated the science in...reasons why this was the first program I used to create graphics for the Addgene blog. However, it has ...requested, although there’s no guarantee Biorender will create it. Additionally, you can upload your own images...

...positive selection, negative selection, and/or screening. We’re focusing on positive and negative selection...in this blog post, but don’t worry, we’ll cover screens in a future post. Positive selection: Cells that...with or without your inserted DNA) and further screening will help you determine if the plasmid backbone...arises in yeast. For example, Markus Ralser’s lab created a yeast prototrophy kit containing 23 plasmids ...bacteria to grow on sucrose. Plasmid cloning: Creativity with naturally occurring genes  At this point... scientists are finding more and more ways to creatively perform selections in plasmid cloning. Additional...

...membrane depolarization increases the BRET signal whereas hyperpolarization decreases the signal. The authors...technologies that can accelerate your research. The creation of NanoLuc® Luciferase from a rather dull 19kDa... by NanoLuc® luciferase (NLuc) and set about to create an in vivo imaging reporter by linking two CyOFP1...-Lanterns with NLuc and called their improved creations Enhanced Nano-Lanterns. These great imaging tools...the two fluorescent proteins together thereby increasing their FRET signal. FRET is a non-radiative transfer...overlap with this optogenetic tool, they needed to create a calcium sensor with the following characteristics...

...International Mentorship Program (IMP) USA-Europe was created. IMP as an idea was conceived and developed by ...the specific technical and logistic support to increase the chances of success, but also nurture a supportive...country of origin with an international mentor creates a closer and more approachable relationship for...at IMP In these sessions, mentors helped mentees create “Career Development Plans” which provided paths...developmental needs and resources needed to pursue them, increased their network of contacts important for their ...Conference highlights included remarks by the Spanish Secretary of Research, Development, and Innovation, D. Carmen... as they witness their student’s progress and increases in self confidence. These improvements, combined...

...CRISPR pooled gRNA libraries that can be used to screen for genes involved in a wide variety of cellular...processes. While these are widely requested and incredibly useful tools, they require extensive background... infect target cells, and, finally, conduct the screen. To help alleviate some of this labor for our ...optimization-scale infection and at least one screening-scale experiment achieving the gRNA representation...) says: "When you're setting up to do a genetic screen, you need to focus on building a really good model...use along with lentiviral packaging plasmids to create your own lentivirus stocks. Our research scientists...

...self-assembling split GFP (GFP1-10 and GFP11) to increase fluorescence 10-fold after protease activity (... (To et al., 2016). However, the lab wanted to increase the fluorescence range to better study the spatio-temporal...β-strand of GFP, hence the name FlipGFP. FlipGFP increases fluorescence 100-fold compared to other reporters...can be applied to other fluorescent proteins to create an array of multicolor reporters that will enable...apoptosis during embryo development. GFP fluorescence increased over time and showed a clear pattern in the developing... This reporter achieves approximately 100-fold increase in fluorescence after cleavage by executioner ...

...Natsume et al., 2016). Now, the Kanemaki lab has created a new toolkit of plasmids for the N- or C- terminal...makes it easy to tag your protein of interest to create conditional human cell lines. Lastly, the Kanemaki...sites adjacent to an NG PAM in human cells. The increased versatility of the engineered SpCas9-NG and its...enhancers, or have attempted to engineer loops to create genomic interactions that wouldn’t normally occur...develop dCas9 for synthetic looping, the Phillips-Cremins lab developed the light-activated-dynamic-looping...were able to observe loop formation as well as increased Zfp462 expression within 4 hours of blue light...

...could mean needing to increase crosslinking to counteract the effects of increased sample agitation; a ...such as qPCR or next-gen sequencing. ChIP is an incredibly useful application for exploring these interactions...crosslinking, which uses formaldehyde or UV light to create strong but reversible bonds. This will keep the... necessary for most ChIP experiences, but the increased binding can mask the epitopes antibodies recognize...shorter sonication time could be an opportunity to decrease crosslinking. (Homogenization for a tissue sample...

...David Liu’s lab created the first base editor in 2016 (Komor et al., 2016) and since then has been trying...David Liu’s lab set out to evolve ABEs with an increased compatibility to Cas homologs. Evolving better...bacteriophage, which rely on the expression of gene III to create infectious particles. A plasmid-based genetic circuit...III to base editor activity and editing speed. Created with BioRender.com. Introducing ABE8e - a fast...about 7 times higher than ABE7.10. However, the increased editing speed and targeting scope of ABE8e comes...comes with a price: ABE8e showed increased off-target DNA and off-target RNA activity. To mitigate this...

...psychological sciences were called into question after credible attempts to replicate major findings could not...our readers into believing the results are more credible than they actually are. Throwing out plates that...preregistration Preregistration is the process of creating a time-stamped research plan in a public repository...the Center for Open Science in order to reward increased transparency and reduced bias in scientific research...Openness Promotion Guidelines; preregistration to increase clarity in study design and analysis; removing...bias with Registered Reports; and recognizing increased transparency with badges. Before coming to the...

...CIB1 to regulate botulinum neurotoxin were also created. The depositors demonstrated the utility of light-mediated...these gas vesicle proteins scatter sound waves and create ultrasound contrast. At a very low volumetric density...s lab at MIT pushed the field further with the creation of the largest miRNA sensor collection reported...al., Nat Commun, 2019. PubMed PMID: 29934631. Screening immune cell lineages in vivo using the CRISPR-...tools have been used in immunology for in vitro screens of immune cell activity. However, a CRISPR-based...gene. CHIME can also be used for high throughput screens using pooled libraries. Find the CHIME plasmids...

...targeted via RNAi. Recently the Vellai lab has created an improved RNAi vector that leads to significantly...non-specific nucleotides into the dsRNA leading to increased RNAi efficiency. Find the T444T plasmid here... used yeast display-based directed evolution to create APEX2. They have now applied the same directed ...the biotin ligase from which BioID was derived, creating a pair of enzymes that overcome both the speed...BioID, which only functions optimally at 37℃. The increased efficiency of TurboID and miniTurbo makes PL a...include protein stability, small molecule binding screens, peptide arrays, and phage display selections. ...

...recording data. Increasing the voltage will shift the population to the right while decreasing the voltage...clearly distinguishable as two separate populations. Created with biorender.com. Pro tip! If your samples...smeary dot plot shows the population of interest. Created with biorender.com.   In such cases, instead...histogram plot for a yes/no flow cytometry readout. Created with biorender.com.   Using histogram plots...baseline expression of the protein of interest. Created with biorender.com. The plot above is for a...

...assaying a secreted protein, first block the Golgi apparatus with Brefeldin A to inhibit secretion and then...through the cell membrane. Formaldehyde fixation creates cross-links between lysine residues thus preserving...flow cytometry data users will typically start by creating a dot plot of FSC versus SSC that allows them ...initially gating the lymphocytes, one could then create a dot plot of CD3 (T cell marker) versus CD19 (...cells, might then gate the B cell population and create a histogram of CD19-containing cells versus total...cells in that sample. Alternatively, they could create additional dot plots of various CD markers to further...

... technology has been used to optimize protein secretion and expression in the biotech workhorse yeast ...Engineering disease resistance through gene stacking, increasing production of a protein of interest, and improving...genetic circuits with multiplexed guides, mini screening libraries, direct edit optimization, and more....can be invaluable for more complex editing and screening experiments in organisms that don’t yet have these...modify it as necessary. And remember, if you end up creating a really cool MoClo kit, we would love for you...Modular Toolkit for Generating Pichia Pastoris Secretion Libraries.” ACS Synthetic Biology, vol. 6, no.... of the GoldenBraid Modular Cloning System and Creation of a Toolkit for the Expression of Heterologous...

...phobias–it’s a key part of being a scientist and an incredibly useful transferable skill. Being able to stand...even in my head. What came out, though, was an incredibly calm: “Excuse me, I seem to have lost my place...any silence, to talk. That moment taught me an incredibly valuable lesson: Your presentation will be better...your slide deck gets uploaded. Your mic emits screeching feedback. You’re muted on Zoom. Your video conferencing...provide quick updates during a company meeting increases visibility of your work, opens up collaboration...Conflict Resolution Transferable Skills Guide: Creativity...

...may not result in any cells, or all the colonies screened might not contain the correct plasmid. There’s...and if the sequence has high GC or AT content, increase the overlapping region of the primer to 40-60 ...buffers used in the previous reactions. This will increase your chances to have a successful and ultimately...pick the big colony and you are safe! Colony screening Pick a few colonies (5-10) and grow them in a ... you've identified potential clones after the screening, sequence the plasmid. Many vectors contain sequences...can use the chromatogram to spot whether any discrepancies between your sequencing result and the expected...