...mutated allele introduced from a conjugative plasmid replaces the chromosomal copy. This is a versatile technique...modifications to pDS132 (Philippe et al., 2004): Replaced the sacB cassette with one of three toxins from... a broad array of pathogenic Proteobacteria. We placed the toxin genes under control of a rhamnose-inducible...the Salis group) (Espah Borujeni et al., 2014). Replaced the pDS132 MCS with a versatile, synthetic MCS... “pTOX” in a broad group of Gram-negative Enterobacteriaceae including in E. coli O157:H7, Shigella flexneri... point mutations. I think it will always have a place in every bacterial geneticist’s toolbox. Depending...negative selection systems for unmodified Enterobacteriaceae." Nucleic acids research 43.13 (2015): e83...

...assembly of biotinylated SpyTag-SpyCatcher linkers and spacer proteins ending with a GFP nanobody (Figure 1)....determination. A) Schematic of workflow. B) Detail of spacer peptides (3HB: 11-nm 3-helix bundle; SAH: 60-nm...software (green circles), magnetic bead (*), and spacers are labeled; scale bar: 100 nm. D) 3D model of ...other targets. The modular system of linkers and spacers could enable many future variations using nanobodies...Cryo-EM samples can be damaged at the air-water interface (AWI) during the freezing process, resulting in...When proteins (blue) interact with the air-water interface (AWI) before vitrification, they can be completely... Small LEA proteins as an effective air-water interface protectant for fragile samples during cryo-EM ...

...new teaching space at Lab Central to learn all about it. LabCentral is a lab and office space shared by ...and making space for the techniques ourselves. As you probably have limited time and space in your own...by numerous emerging biotech companies. The space was modern with clean bold colors of orange and white...like workshops at Lab Central. Because of the fast pace of work in the Addgene lab, we don’t often get the...opportunity to explore new techniques in our own space. Workshops such as this give us a chance to learn...

...many histidine residues to include.  Placement As far as placement goes, there is no cut or dried answer...frequency amino acid, proteins containing two or more adjacent histidines are present in most production systems...polyhistidine tag plasmids here! Where should I place the tag and how many do I need? When designing a...ions once the protein is folded. Sometimes a tag placed on one end will be blocked during protein folding...should try tagging the opposite end.  If neither placement works, consider purifying under denaturing conditions...terminus is preferable over the other however, tag placement can affect protein function. B, The string of ...

...receptors expressed on the cell surface. Thus, a screen can be used to target surface proteins to determine which...known to cause the up-regulation of an important surface protein (e.g. PD-L1 on a cancer cell). In this ...signaling molecules were involved in the phenotype. Surface proteins  My group works on immune cells in different...transferred cells is followed. A benefit of working with surface proteins is also that such proteins are easily ...further be used to interfere with the identified surface proteins, and such antibodies could thus be interesting...all differentially expressed genes that code for surface proteins. One of the first challenges with a custom...the information that you have input in the web interface, making it convenient for you to later identify...

... B cells. They bind to proteins on the surface of extracellular pathogens such as parasites or microbes...microbes, or to proteins expressed on the surface of cells that have been infected with a microbe, to trigger...the amount of protein present. The reaction takes place in a 96-well plate, where primary and secondary ...Flow cytometry is used to detect proteins on the surface of or inside whole cells. Antibodies - typically...cells: antibodies can bind to proteins on the cell surface, or the cell membrane can be permeabilized to allow... allow antibodies to bind to intracellular proteins. The fluorescence signal is detected by the flow cytometer... activate cellular pathways by binding to cell surface receptors. These applications are typically used...

...LanthaScreen) (Degorce et al., 2009), measuring intracellular calcium (Miyawaki, Griesbeck, Heim, & Tsien,... to get your fluorophore delivered to the right place? FRET experiments require targeting the donor and...specificity Donor fluorophores need to go to the right place. Mis-targeted donors never “see” FRET acceptors,... site when used in conjunction with Cy dye-nitrilotriacetic acid (NTA) conjugates (Kapanidis, Ebright,...proteins (such as GPCRs, ion channels, or cell surface receptors) or with experiments on permeabilized...Biarsenical labeling reagents If you want to label intracellular proteins covalently with FRET acceptors, then...and the acceptor fluorophore binding sites are adjacent to each other on the same protein and a negative...

...transsynaptic neuronal tracer into a monosynaptic (first order) neuronal tracer For RABV to crawl through...neurons are connected. Viral Vectors as Neuronal Tracers Due to their ability to efficiently enter cells...However, they generally are not transmitted to adjacent neurons via synapses (i.e., transmitted transsynaptically...cells through their interactions with target cell surface receptors (Callaway 2008). Based on this, cell ... RABV expressing the viral protein EnvA on its surface will only infect Cre-positive, TVA-expressing cells...taken up by the presynaptic nerve terminals of adjacent neurons (i.e., it is taken up by projection neurons...

...better access to intracellular targets. If your target is already on the extracellular side of the membrane...proteins in place, while allowing antibodies into the cell where they could bind to intracellular epitopes... Organic Solvents Organic solvents, generally acetone, methanol, or ethanol, work by dehydrating and ...and leaves many membrane-associated proteins in place. In contrast, Triton X-100 and Tween-20 are examples...is suitable for antibodies that bind to the extracellular portion of a protein. B) Permeabilizing with...

...Lab: Classic Edition Winners In a tie for first place, the Vestibular Lab decorated not just their lab...@druryhannah) The Tarling-Vallim Lab’s first place-winning entry doubled as their lab’s decoration ...Lab (@TarlingVallim) December 19, 2020 In third place, the BTC Institute decorated with dozens of individual...Deck the Lab: Home Edition Winners Winning first place in the home edition, the MicrobiGals created glow...Microbigals (@MicrobiGals) December 23, 2020   In second place, Dr. Anne Madden created a microbe advent tree displaying...Madden (@AnneAMadden) December 23, 2020   In third place is Matthijs Dorst’s Deck the Lab Nativity scene ...

...Penguin Publishing Group. Day, F. (2019). Embrace your weird face your fears and unleash creativity. Gallery...focus. Find yourself a block of time and a quiet space where you can address the issue head on. Prepare...can come any time. Meetings shouldn’t be the only place people share ideas. Ideas had over lunch are just.... Identify your enemies In Felicia Day’s book Embrace your Weird, Day identifies nine enemies that stand...backordered for months, we have to have plans in place to be able to continue our experiments. Thinking...team has used these techniques to optimize our lab space, our protocols and even our Lab Inventory Management...

...the sequence is located just upstream of a Protospacer Adjacent Motif (PAM sequence). The 3-5 nucleotide...modifications at DNA targets adjacent to NGG PAM sequences when used in place of wild-type SpCas9. The remaining...enzymes require a single base protospacer flanking sequence (PFS) adjacent to the target, but many do not... REPAIR (RNA Editing for Programmable A to I Replacement) system fuses Cas13b to RNA deaminase ADAR2 to...

...paragraph is the place to introduce yourself and your background. It’s a great place to state why you’...What is a cover letter for? A cover letter is a place to provide helpful context about your candidacy,...candidacy, beyond what is on your resume. It is a space for you to explain how your skills and experience match...candidate!  Additional uses The cover letter is a great place to proactively address questions about your application...you haven't talked about it yet, it's also a good place to state why you are interested in working for this...applicants. In short, while a cover letter can’t replace skills and experience, it can help the hiring manager...

...Bordetella spp., Caulobacter spp., Enterobacteriaceae, Gluconacetobacter xylinus, Haemophilus influenzae,...Gram-negative and Gram-positive  Acinetobacter calcoaceticus, Actinobacillus pleuropneumoniae, Actinomyces...carotovora, E. chrysanthemi, Escherichia coli, Gluconacetobacter xylinus, Gluconobacter spp., Hyphomicrobium...pSa, pR388 Gram-negative  Acinetobacter calcoaceticus, Aeromonas liquefaciens, A. salmonicida, Agrobacterium...campestris pv. campestris, X. campestris pv. malvacearum, Zymomonas mobilis pBBR1 based pBBR1 ...Caulobacter crescentus, Escherichia coli, Gluconacetobacter xylinus, Paracoccous denitrificans, Pseudomonas...

... Angela DePace has put personal development at the center of her lab (pictured above). DePace holds yearly...through thoughtful planning and honest feedback DePace’s yearly planning meetings revolve around two simple...tone of the meeting is almost more important. As DePace says, science is full of “terrible perfectionists...have tangible benefits for both PI and trainee. DePace and her lab members have noticed that the meetings... Park J, Bragdon MD, Lopez Rivera F, Biette KM, DePace AH. Mol Cell. 2015 Jun 4;58(5):718-21. doi: 10.1016...

... in the extracellular domain which activates the native signaling cascade of the intracellular part of...ligands (PORTLs). OptoXRs are chimeras of the extracellular domain of endogenous light-sensitive receptors...receptors, like mammalian rhodopsin, and the intracellular domain of an endogenous GPCR, such as adrenergic...signaling pathways? Here’s where photoswitchable intracellular proteins come into play. Photoswitches can be...state.      Figure 4: Example of an intracellular signaling pathway activated by an optogenetic...., & Cui, B. (2015). Optogenetic control of intracellular signaling pathways. Trends in Biotechnology,...

...rich and diverse workforce. Out of all the career spaces I explored for my post-PhD life, the science policy...science policy. Luckily, there are many mechanisms in place to ensure your effective networking---and they’re...pandemic has foisted upon us. Action Items:  A simple place to look for science policy folks is on Twitter- ...! Someone from your alumni community is a great place to start. What is science policy? THE IMPOSSIBLE... policy is so diverse, you can do it in so many places-- not just the government. When you talk to folks...technologies accessible to patients. And what better place to do that than at the nation’s largest integrated...to chat with someone working in a science policy space you’re interested in. Many fellowship programs can...

... entered human cells via hACE2, could not enter cells expressing murine ACE2 (Subbarao et al., 2006). ...SARS-CoV-2. The resurgence in popularity of the hACE2 transgenic mouse strain, for example, is sustained...the University of Iowa (McCray et al., 2006), the hACE2 transgenic mice were designed after it was discovered...Repositories around the world facilitate the rapid pace of SARS-CoV-2 research by removing logistic hurdles...Look DC, Perlman S (2006) Lethal Infection of K18-hACE2 Mice Infected with Severe Acute Respiratory Syndrome...

... that helps attack extracellular pathogens.  Transportation of antibodies to places they can’t reach on...valency of 10 antigens. An extra constant domain replaces the hinge region in IgM antibodies. IgM has no.... IgD While IgD is coexpressed with IgM on the surface of most immature B cells, its function is unknown.... They are found bound to IgE receptors on the surface of basophils and mast cells, two types of white... the intervening DNA sequence is deleted, which places the variable region next to a different constant...

...target proteins. The surface-exposed loops of nanobodies seemed like a good place to put AsLOV2 because...inserted AsLOV2 into conserved sites in all eight surface-exposed loops of an mCherry nanobody. Only insertion...-dependent shift in mCherry fluorescent to the surface of the bead, suggesting that OptoNBs still bound...bound their target protein even when stuck to the surface of a bead.  Using OptoNBs to control cell signaling...With darkness, SOS binds Ras and initiates the extracellular signaling-regulated kinase (ERK) signaling cascade...