...range between 0 and 100) are then converted to a weight (which ranges between 0 and 1) so that they can...ranging from -40 bp to 40 bp) are also converted to a weight (which ranges between 0 and 1) (shown in the graph...Conclusion CrispyCrunch is now publicly-available for high-throughput design and analysis of CRISPR experiments...

...oneself that prompted us to create Crowdfight.   Figure 1: Crowdfight is a community of scientists who...making science.   How we started Crowdfight-COVID19 Crowdfight was born as a collaborative platform...collaborator for a specific task, Crowdfight will: 1) Find an expert with the right skill; 2) Confirm the expert...Ignacio Amigo and María Hernández Sánchez from Crowdfight. In March 2020, while most of the world was locked...did not have access to a P3 laboratory (BSL-3), a high-security facility needed to perform the experiments... them heard about our online platform, called Crowdfight, and filed a request for help. In just a few ... a research group in Scotland, who had just the right infrastructure and offered to collaborate with them...

...hand, there are reasons why grad school might not be the right choice. I spoke with our Financial Operations...here. You also don’t have to go back to school right away. Shannon Rinaldi, Addgene’s Lab Manager, was.... I thought, ‘Let me pursue this, let me make a slight change so I can have flexibility and be up to date...biggest takeaways from my co-op. It has been so insightful to learn about different career paths, successes...

...and wrap the column with blue LED lights. When you switch off the lights, OptoNBs bind YFP and non-specific... the blue lights. Which option do you choose?  Option #2 may sound like fiction, but light-controlled...dark-induced dark-induced Loop 6 blue-light induced dark-induced blue-light induced Table 1: Summary of the... effect of AsLOV2 insertion site on light-induced binding.  Light controlled protein purification with...while cycling between dark and blue light which revealed a blue light-dependent shift in mCherry fluorescent...cells: light-based control of signaling pathways. This is particularly exciting because using light to control...binding of their target protein is altered by blue light illumination. This ability to reversibly bind and...

...engineering of fusion proteins is relatively straightforward. However, generating a fusion while keeping...discuss strategies to generate fusion proteins and highlight some aspects of their design.  Protein size and...situation Next to size, potential homodimerization and higher order oligomerization of fluorescent proteins is... the fluorescent tag should operate as an inert light emitting module, any tendency of GFP to homodimerize...fluorescent protein characteristics such as the brightness, photostability, maturation time, and acid sensitivity...termini of your protein of interest The most straightforward fusion protein is made by connecting the N-...the fluorescent protein-POI fusions, we generate straight fusions, without any linker. When both orientations...

...the challenge of trying to figure out what system might work best for your particular purposes. We’re here...than others. In today’s post, we will focus on a high-level comparison of popular cloning methodologies...results. If you’re in a hurry, Golden Gate cloning might be for you. This methodology relies on versatile... in as little as 30 minutes! Alternatively, you might consider Gibson cloning. In this technique, you ...fragments in an order determined by their homology. You might also consider FastCloning, first described by Chaokun...approach should you use if the budget is particularly tight? Most cloning processes will require a PCR reaction... job done, provided that you’ve already got the right enzymes laying around the lab. If not, you’ll be...

...conducted in Professor Cepko's lab and to get some insight into her management and mentoring styles. If you'd...

... focused on three different levels of assembly: High efficiency and accurate single-insert assembly Intermediate...destination plasmid. Antibiotic selection allowed for high throughput cloning efficiency measurements. We performed...confirmed the insertion of the Lambda insert at the same high frequencies as the antibiotic selection markers....lacZ cassette assembly was easily achievable with high levels of transformants and low backgrounds – so...Using these 4 bp overhang observations, accurate “high-fidelity” junction sets for both the 12- and 24-...complex assemblies were performed. It was found that high efficiencies and accurate assembly levels were indeed...in the number of transformants while maintaining high levels of correct assemblies) translate to faster...

... (blue light-inducible AraC dimers in E. coli) that turns expression on and off with blue light.   Inspired...domain with VVD, a light-responsive domain that dimerizes upon exposure to blue light (Fig. 1). Another...VenturaIf you follow Barbara Di Ventura on Twitter, you might have seen the video of her singing about the paper...Another blue light-inducible dimerization domain, Vaucheria frigida aureochrome1 (VfAu1), led to functional...most applications.   Figure 1: With blue light, the VVD domains of the BLADE dimerize and activates...under the pBAD promoter, can be controlled with light without the need of any cloning.     What advantages...BLADE activates transcription with the flick of a light switch and shuts off just as quickly. The original...

...This is where FLEx enters the scene. FLEx: A light when night is about you! The FLEx (for flip-excision)...optogenetics? Optogenetics: Controlling neurons with light In 2005, a team from Stanford University, led by...ago with the discovery of microbial rhodopsins, light-sensitive proteins that absorb photons for energy...maintain membrane potential in response to visible light (Zhang et al., 2011). What do microbial rhodopsins...2000). Most neurons in the brain are not naturally light-sensitive, so the selective expression of opsin ...Optogenetic neuromodulation is made possible by the light: when hit with the correct wavelength, microbial..., 2011; Mei and Zhang, 2012). For example, blue light activates channelrhodopsins that, in turn, trigger...

...Scientist. I used to spend my days (and often my nights :D) engineering E. coli to produce biofuels in ... screaming from the lab like the cartoon on the right?!? I would love to tell such a dramatic story, but...This may come as somewhat of surprise to many of my high school friends. In my teenage years I was super ...can involve walking alongside it and giving it a high five. While screaming can be therapeutic and I’ve...

...showed that improved specificity of Cas14 enables high-fidelity SNP genotyping. The development of  Cas14...

...coronavirus using CRISPR-based SHERLOCK (Specific ​H​igh ​s​ensitivity ​E​nzymatic ​R​eporter un​LOCK​ing)... under either no excitation light, UV light or LED blue light.  Highlights: This is a one-pot, one-temperature...heat block, LED blue light illuminator or UV light illuminator. Approach: Highly similar to the SHERLOCK...captured by an antibody further along on the strip. Highlights: Of the 83 known COVID-positive samples tested... extraction but that may work only if there is a high enough (105 copies per microliter) viral load in...lateral-flow readout slip that allows rapid detection.  Highlights: Cas13a is not catalytically active in the presence... duplex and will not give a positive result for highly similar viruses. Unlike Cas12a, Cas13a does not...

...limit of a light microscope. Due to the wave nature of light, an infinitely small point of light will blur... to provide high 3D spatial resolution while also avoiding the toxic effects of high light doses to the...fluorescence microscopy provides a high contrast image in which a bright signal overlays a perfectly black...placed to only allow light from the focal plane to reach the detector and all light from above or below...for example, red light instead of blue to excite a molecule of GFP. Because red light has approximately...approximately half the energy of blue light, two photons of red light are needed to excite GFP compared to only ...objective can a a high enough density of red-photons be establish for this to happen.Using red light has a second...

...UV-Violet Dark/Green N/A (high) High 503/ 518 Padron Monomer Blue Dark/ Green N/A (high) Low 503/ 518 rsCherry...specific wavelength of light. PA-FPs can be activated from low fluorescent states to high fluorescence states...fluorescence, to a brighter fluorescent state by exposure to a specific wavelength of light. The switch or...fluorescence when illuminated with 488nm light. However, when UV-violet light is presented first, it causes irreversible...illuminated with violet light, both variants are photoconverted to forms that emit brighter red fluorescence....when exposed to blue light, which is less phototoxic to live tissue than the UV light used by other PA-FPs...State Activating Light Pre/ Post Color Contrast (Fold Change in Fluorescence) Brightness Excitation/ Emission...

...with exquisite precision using only light. Optogenetics, or light activated tools, can control everything...an experimental optogenetic setup. A light source delivers light pulses to the subject/cells expressing... photoswitches. Light-gated ion channels directly open and close when exposed to light and change the ...changes with light stimulation to either activate or inhibit a specific receptor or pathway.  Light-gated ion...turn on with a light-pulse at one wavelength and stay on until a second pulse of light, with a different...pathways with light. This is achieved by coupling endogenous signaling molecules with light-activated partners... intensities of light sources used to prevent activation of both tools with each light source used.   ...

... to their brightness and high degree of overlap. Dyes for DNA/RNA staining should have high affinity and...refers to the emission of light by a substance (fluorophore) that has absorbed light or electromagnetic radiation...whereby excitation of blue light will indicate where FITC is bound through green light emission. Key considerations...also vary in their brightness, depending on the respective fluorochrome. Brightness is impacted by both...of findings. If you’re struggling to select the right fluorescent tools, this blog is here to help. Here...ligand to make your selection easier, as well as highlight some recent advancements within this field. Fundamental...wavelength (Heinrichs, 2009). When a fluorophore absorbs light, electrons are elevated from a ground state to an...

...neuroscience. In fact, precise light-based control of biological processes is highly useful in other fields, ...consistent, and controllable pulses of light. Complicated patterns of light intensity and wavelength can be ...to low-intensity light, so off-target effects should be limited. Phytochromes are light-responsive systems...harness the power of light, Christopher Voigt’s lab created the first E. coli light-sensitive two-component...construct senses light, and an obligate chromophore allows the system to respond to various light inputs. In...various light inputs, and an obligate chromophore allows the system to respond. In far-red light or dark...exposure to red light rapidly deactivates the system. Version 1 of this far-red/red light-responsive system...

...LACE system for light-activated CRISPR-Cas9 effector, Polstein and Gersbach fused the light-inducible protein...binding affinity with light stimulation2. The Kuhlman lab created two improved Light Inducible Dimers (iLID...of Arch mutants screening for improved brightness. The brightest mutants were then further mutagenized ...enhanced sdChr construct, named CheRiff, exhibits high light sensitivity and fast kinetics. By combining both... weak or high activities (Bert, et al. 2000). To design pXPG, the authors incorporated a high-copy origin...repository, we can't give all of them attention they righlty deserve, but, in this post, we'll provide a small...2015 Feb 2;33(2):139-42. doi: 10.1038/nbt.3149. Light-inducible CRISPR-Cas9 system for endogenous gene...

...produce the same amount of light from an Fluc or RLuc reaction. In fact, NLuc is bright enough to detect endogenous...require external light sources. Unfortunately, detecting traditional luciferases’ dim light emissions requires...reporters produced bright signals without exogenous excitation and with OgNLuc being the brightest since it had...mutations and 2 deletions. CyOFP1 has a higher quantum yield, brightness and improved maturation time compared...Nano-Lanterns. RLuc8 is 5.5-fold brighter than RLuc but NLuc is 30-fold brighter than RLuc8 (6). Susuki, K, ... characteristics: No photoexcitation High Ion specificity High sensitivity Easy expression Gives ratiometric...coelenterzine substrate to the more stable yet brighter furimazine by Promega’s Chemistry group is a good...