...Fellow in the Church and Collins labs at the Wyss institute for Biologically Inspired Engineering. Alex is...Listen to this episode for insights into the process of getting an MD PhD, the inside scoop on developing new...postdoc 12:01 - 13:21 : The Wyss Institute for Biologically Inspired Engineering and why Alex decided ...mutant microbial strains faster than ever before. 29:54 - 31:25 : Nature's defenses against gene drives. ...Page. 2:00 - 3:55 : Alex introduces himself and explains why he decided to do an MD PhD - it's all in the...- 7:04 : Alex describes the MD PhD process and explains why he loved the first 2 years of medical school.... 13:22 - 22:07 : Alex's projects at the Wyss Institute - New CRISPR tools and the importance of moving...

...binding proteins in as little as 3 days. Abil et al., 2014 show that you can fuse custom PUF proteins to a...you’re an academic researcher at a nonprofit institution, you can order plasmids covering a wide range...expression in bacteria, yeast, mammalian cells, insects, and even Xenopus oocytes. The expression vectors...antibiotic efflux pumps and ankyrin seven-repeat proteins: Sjuts, Hanno, et al. "Molecular basis for inhibition... from affecting its translation initiation and instead makes initiation heavily dependent upon the core...Biosafety of biotechnologically important microalgae: intrinsic suicide switch implementation in cyanobacterium... "Modular Synthetic Inverters from Zinc Finger Proteins and Small RNAs." PloS one 11.2 (2016): e0149483...

...with dimerizing fluorescent proteins is illustrated. 1) Membrane proteins fused to FPs that dimerize can... National Institutes of Health and he is currently an Associate Professor at Albert Einstein College of...-based FRET, with application to intrinsically unstructured proteins. Protein Sci, 2007. 16(7): p. 1429... Erik L. Snapp. Stop using EGFP/GFP for fusion proteins! Despite multiple studies in high profile journal...(i.e. DsRed) [3]. This is a problem for fusion proteins. One of the major applications of FPs is to visualize...Transmembrane fluorescent protein fusions Transmembrane proteins, i.e. receptors and transporters, are integrated... to an obligate dimer or oligomer Several cell proteins normally self-associate into homodimers or even...

...sorting or FACS (2). By analyzing single cells instead of the population as a whole, scientists gain statistical...Techniques is Best for Me?  An overview of the instrumentation A flow cytometer combines three systems to ...receptor are stained with a fluorescent antibody against the receptor and analyzed on a flow cytometer. ...cells pass through the path of a laser and the instrument collects information about the cell’s size, complexity...targets. If you are planning on using fluorescent proteins for your experiment and are unsure which to choose...Fluorescent Protein Should I Use? However, fusion proteins tag your protein of interest with a fluorescent...C-terminus of the protein. One downside of fusion proteins is that the tag can alter the structure of the...

... Georgeta, and Justin W. Taraska. "Imaging proteins inside cells with fluorescent tags." Trends in biotechnology...Janelia Research Campus, Howard Hughes Medical Institute. Chemistry is dead, long live chemistry! The discovery... their own. Subsequent advances in fluorescent proteins have replicated many of the properties once exclusive...budgets are a widespread issue with fluorescent proteins—even the thriftiest cell biologist can feel like...brighter and more photostable than fluorescent proteins, providing a straightforward way to improve the...combined with the ease and specificity of fluorescent proteins. In-cell labeling strategies The majority of ...tetracysteine (Cys4) peptide tag could be used to label proteins in cells (Figure 1a). Other strategies that have...

...occurring E. coli plasmid ColE1 (Goebel & Helinski, 1968; Bazaral & Helinski, 1968), in which the plasmid was ...1981). E. coli strains such as DH5α, HB101, NEB Stable, Stbl3, etc., are recA- mutant strains, and therefore... of plasmids, taking many forms (Levene, 2009; Higgins & Vologodskii, 2016). Plasmid multimerization occurs...to occur more frequently in plasmids with large inserts (>7 kb)(Berg et al., 1989), direct repeats (Ribeiro...et al., 2009). Since multimers have additional origins of replication compared to monomers, they are also... of multimer present in your sample. In these instances, please note that the supercoiled dimer band can.... If the presence of a multimer in your sample remains unclear after visualizing it on a gel, one of the...

...Mutations Insertions, deletions, point mutations Insertions, deletions, point mutations Insertions, deletions...-4 phage proteins are needed and these can be provided via a separate plasmid or be inserted into the ...recombineering proteins Red/ET from a temperature-sensitive plasmid. Homologous recombination then joins the linear...successfully incorporated a 60-nucleotide insertion; beyond this insertion size, primer synthesis is more expensive...making small point mutations, large deletions/insertions require complicated, often costly in vitro assembly...the power of bacterial recombineering to create insertions, deletions, and substitutions - at the same efficiency...colonies carrying the unmodified plasmid. For insertions, it’s possible to use the same technique just...

...REC and NUC, and their subdomains. NUC includes the HNH and RuvC catalytic domains, as well as the CTD (...recognition). The NUC lobe contains the DNA scissors: the HNH and RuvC domains, which are named for similar...the guide RNA. Unlike the cleavage domains, the recognition domains—including the CTD and the entire REC...similar nuclease folds found in other proteins. The C-terminal domain (CTD) of the protein also belongs ...lobe—do not structurally resemble any other known proteins; they are completely unique to the CRISPR system...Nishimasu et al., 2014). Table 1. Summary of Cas9 domains, the lobe each domain belongs to, and their roles...pyogenes Cas9 in the apo state (PDB ID 4CMP) with domains colored as in Figure 1. HNH, red; RuvC, orange;...

...binding of untagged proteins. Despite being a relatively low frequency amino acid, proteins containing two ...research requires a close examination of specific proteins within a system. This can be pretty complicated...given that a single cell has tens of thousands of proteins functioning in a variety of ways. How do scientists...from a complex mix using affinity chromatography against an amino acid sequence, or tag, on the protein....of methods can be used to successfully isolate proteins, this blog will focus on one of the most common... as Ni2+ or Co2+ (Hochuli 1988), and can elute proteins with up to 95% purity (Janknecht 1991, Hochuli...itself. Next, a series of wash steps removes excess proteins and other contaminants. The protein of interest...

... and analysing the insecticidal potential of Cry proteins in order to manage insect pests and to support...The recombinant Cry proteins will then be screened for toxicity against local insect pests. By developing...the identification of plant compounds and insecticidal proteins to the development of renewable biotechnology...desaturating fatty acids. Identification of Insecticidal Proteins Linh Bao Ton (M. Sc.), Nong Lam University...developing biological agents such as insecticidal proteins, the use of chemical pesticides may be avoided or...system pDawn to clone and express newly identified proteins and enzymes. These compounds will later get tested... nutritionally and therapeutically significant proteins. In this project she aims to create transgenic...

... of proteins captured on magnetic beads (Arimura et al., 2024). In MagIC-cryo-EM, target proteins (expressed...adding Late Embryogenesis Abundant (LEA) proteins, small proteins that naturally adsorb to the AWI, can ...editor installs the recombinase “landing sites,” then the recombinase handles the installation of the ... DNA-targeting proteins, so the team created versions using dCas9, zinc finger proteins, and transcription...for enrichment and structure determination LEA proteins protect samples during cryo-EM grid freezing Engineered...biotinylated SpyTag-SpyCatcher linkers and spacer proteins ending with a GFP nanobody (Figure 1). This enrichment...https://doi.org/10.1101/2024.01.21.576499.    LEA proteins protect fragile samples during cryo-EM grid plunge...

...Broad Institute focuses on proteomics: developing and applying new technologies to quantify proteins in ...locus and labels the proteins in its vicinity with biotin. The biotin-labeled proteins can then be isolated...electron-rich amino acid side chains (e.g. Tyrosine) with biotin. These biotin-labeled proteins can them be analyzed... anti-GFP nanobody is able to bind fluorescent proteins derived from A. victoria the functionalized nanobodies...: 29915061.        A new method to identify proteins occupying a specific genomic locus: Caspex  Article...group, developed a CRISPR-based approach to detect proteins that associate with specific genomic loci. He ...engineered peroxidase, (APEX) that labels proximal proteins with biotin as an affinity tag. This ‘Caspex’ ...

... Chavez, researchers at the Wyss Institute for Biologically Inspired Engineering. CRISPR/Cas9 technology...to effector domains such as transcriptional activators or repressors. These fusion proteins retain the .... For instance, when native (nuclease-competent) Cas9 interacts with a ≤15nt gRNA that contains an RNA..., or guide RNA (gRNA). The gRNA conventionally contains a 20-nucleotide sequence that is complementary..., in the genome. Native Cas9 has two catalytic domains, each of which cleaves one strand of DNA upon binding... full-length gRNA, native Cas9's two catalytic domains, HNH and RuvC, cleave DNA strands complementary... a regulator of gene expression. Its catalytic domains can be mutated to inactivate the protein’s nucleolytic...

... measurement that is unaffected by intervening proteins, lipid bilayers, organelles or other cellular ...et al., 2007). All that is required is that the proteins/nucleic acids/lipids of interest are fluorophore...resulting from nonspecific donor labeling. Fluorescent proteins (FPs) are the gold standard for genetically-encoded...remember that, if you make an FP fusion, you are inserting about 20-25 kDa of protein mass into your protein...protein and so structural changes due to this bulky insertion are inevitable. Most FP fusions for FRET studies...can affect the structure or function of certain proteins. Rather than shove a bulky FP into your protein...pair, a researcher can lure the ribosome into inserting a fluorophore-tagged amino acid at a stop codon...

... Vector Database. Step 2: Insert name  Include information about the insert in your plasmid name. This...position to the insert. Example, if you have a Flag tag on the N-terminal of your insert, you would list...fused to the C-terminal of your insert you would list it after the insert. pBACKBONE-Flag-hGene-EGFP Other...Other considerations  If your insert contains a mutation or modification, this should be included in the...versions of the insert are often denoted by "wt" either directly before or directly after the insert name.   ... quickly and easily identify what your plasmid contains and other important information. Tip: A lowercase...antibiotic resistance, b) the promoter that drives the insert, and c) any other selection markers (for use in...

...Zhou: Fluorescent Light-inducible Proteins (FLiPs) Fluorescent proteins can be applied in a long list of...light. To do so, she generated fusion proteins in which Dronpa domains flanked either side of an enzyme’s...Fluorescent proteins have enabled scientists to pursue creative research avenues previously unavailable...researchers are finding new ways to apply fluorescent proteins everyday.  The late Michael Davidson and Roger...for their creativity in the use of fluorescent proteins and the potential for their work to enable future...that list by using photoswitchable fluorescent proteins to create enzymes that can be turned on and off... exposure caused oligomerization of the Dronpa domains thereby occluding the enzyme active site and shutting...

...for tDNA insertions into the plant’s germline Selecting for seeds that have the tDNA insertions (usually...have no control over how many insertions occurred. Having too many insertions can cause artifacts from over-expression...control over where your construct will be inserted. If your insertion occurs in a coding region, it may cause...transcriptional and translational dynamics of genes and proteins in living plants, to complementing mutant phenotypes... process occurs in nature; Agrobacterium tDNA insertion likely precipitated domestication of sweet potatoes...and generating N and C terminally tagged fusion proteins with fluorescent (YFP, GFP, CFP, RFP, etc.) or...perform the transformation not long after flowering begins. If your plants begin to flower before your constructs...

...only indicates the presence of AN insert, not necessarily YOUR insert. Any cloning artifact that disrupts...able to choose those that contain plasmid with an insert? Blue-white selection is a widely used method to...beginning. The well-characterized bacterial lac operon contains a gene called lacZ that encodes for the enzyme...indolyl-β-D-galacto-pyranoside) into galactose and an insoluble blue pigment (4-chloro-3-brom-indigo). So far,...an orange wedge in the figure on the left) and inserted it into a plasmid, creating an α-complementation...representative plate to the right, colonies with an insert-containing plasmid have a non-functional β-galactosidase...control: Transform the backbone plasmid without insert. All colonies on this plate should be blue, indicating...

...Microbial rhodopsins are encoded by opsin genes, and include bacteriorhodopsins, halorhodopsins, and channelrhodopsins...of optogenetics begins almost 50 years ago with the discovery of microbial rhodopsins, light-sensitive...light-sensitive proteins that absorb photons for energy conversion or the induction of intra- or intercellular signaling... et al., 1984; Harz and Hegemann, 1991). These proteins control the flow of electric charges across cell...light (Zhang et al., 2011). What do microbial rhodopsins and neurons have in common? Neuronal stimulations...when hit with the correct wavelength, microbial rhodopsins enable ions to flow across the axon’s membrane... 2012). For example, blue light activates channelrhodopsins that, in turn, trigger neural excitation, ...

...system is likely applicable to many types of proteins. For proteins with a modular structure, constructing ...Other proteins may also be utliized as scaffolds, including the set of red fluorescent proteins. The T-DOGGs...researcher Jonathan Tang wanted to express a variety of proteins in single cell types in the mouse, but knew it...single heavy chain, and they are small and stable inside cells. Nanobodies binding GFP were designed in ...showed that nanobodies could be fused to other proteins, and these fusions retained the ability to bind... Tang et al. tested pairs of their GFP-binding proteins (GBPs) to find those that could co-occupy GFP....similarly to GFP, but commonly used red fluorescent proteins dsRed, mCherry, and TdTomato do not induce transcription...