...executive director of Central Indiana Science Outreach and a Postdoc at Indiana University School of Medicine...instance, I tweeted to the Indiana State Museum and others when I was trying to find local informal science... mine was geared toward careers in academia or industry. Luckily, I discovered that there are many ways...culminated in my founding a nonprofit called Central Indiana Science Outreach, or CINSO. We organize fun science... you’re provided throughout your PhD program.   Find people who will support your professional development...advisors are not as supportive of your career goals, find people who are. I wanted to do fun science activities...

...Resources on Addgene.org Find Cre-Lox Plasmids Find Optogenetics Plasmids Find Chemogenetics Plasmids ...recombinase together with inducible Cre recombinase (CreERT2), visualizes individual neurons and their synapses...but share an overall structure of 13 base pair palindromic repeats separated by an 8 bp asymmetric core.... recombinase efficiency are important to keep in mind when deciding whether to use the Cre-lox or one ...compatible target sites are cleaved out, leaving behind a vector with the inverted DNA flanked by non-compatible...networks Double inverted vectors expressing light inducible ion channels (plasmid # 20298 for example) have...to fuse a mutant estrogen receptor (ER) ligand binding domain (LDB) to the C-terminus of FLP or Cre (pCAG-CreERT2...

... does inducible expression work? As mentioned above, many expression plasmids utilize inducible promoters...'inactive' until an inducer such as IPTG is added to the growth medium. Induction timing is important,...long to induce, your culture will start collecting dead cells, and, conversely, you cannot induce too early...compatible. An arabinose-inducible plasmid will not express in an IPTG induction strain for example, nor...engineered to accomodate high protein loads using inducible promoter systems (which will be discussed later...Features Genotype Use BL21 (DE3)   Basic IPTG-inducible strain containing T7 RNAP (DE3) F- ompT lon hsdSB...toxic proteins BL21-A1 Tetracycline Arabinose-inducible expression of T7 RNAP; IPTG may still be required...

... lower IREB2 binding enrichment compared to wild-type sequences and that loss of binding correlated with...with Split-BioID Find resources for studying chromatin Resources on Addgene.org Find Split-BioID plasmids...plasmids Find CRISPR plasmids for labeling genomic regions Find CRISPR plasmids for purifying specific genomic...characterization of RNA-protein interactions has lagged behind, likely due to limitations of current means to ...and allows a researcher to identify proteins that bind an RNA motif of interest in living cells. Challenges... has limited utility for identifying novel RNA-binding proteins that interact with an RNA of interest....distinguish between proteins that directly or indirectly interact with the bait. Also, RaPID can’t be ...

...antibodies to bind. We also know that changing the Fc domain can impact an antibody's binding affinity (Janda...express Fc receptors that bind to the Fc domain of an antigen-bound antibody and induce (or, in the case of ...interact with, so IgG isotypes bind to Fcγ receptors; IgE isotypes bind to Fcε receptors; and so on and...antibodies bind to a pathogen and natural killer (NK) cells expressing Fcγ receptors binds to IgG and ...know much about yet. Who knows what kinds of functions they may induce?  Fc receptors are intriguing from...system, including the innate immune response, by binding to Fc receptors on other immune cells. (psst! Need...Antibody-dependent complement deposition (ADCD) Antibodies can bind to a pathogen and physically prevent it from entering...

...(470nm), paCas9-1 induced indel mutations via NHEJ (frequency of 20.5%) and induced modifications by HDR...the light-inducible heterodimerizing cryptochrome 2 (CRY2) and calcium and integrin-binding protein 1 ...background and high fold-induction of Cas9 activity (16.4-fold). This paCas9-1 light-inducible system was able ...flanking the DNA-binding cleft of Cas9, the fluorescent domains dimerize and prevent DNA binding in the dark...Upon expression in the cell, the dCas9-CIB1 fusion binds to the target DNA sequence as directed by the guide...background activity in the dark state and highest fold induction at 31X. By using a slit pattern during blue light...domain VP64. Using this system in HEK293T cells to induce expression of human IL1RN, the researchers saw ...

...primer binding site, starting with a length of about 13 nt (Anzalone et al., 2019). Primer binding sites...designed to bind only after the edit is installed. This reduces concurrent nicks, which lowers indel rates ...the primer binding site (Nelson et al., 2022). PE7: This editor includes the fused RNA-binding protein La... of a CRISPR gRNA extended to include a primer binding site (PBS) complementary to the genome and a reverse...Starting lengths for optimizing your pegRNA primer binding site (PBS) and reverse transcriptase template (...), disrupting its canonical structure and Cas9 binding (Nishimasu et al., 2014). Figure 3: pegRNAs...while pegRNAs that use other bases in this location bind to Cas9 with canonical sgRNA structure (right). ...

...reinforced by the findings of the 1952 Hershey-Chase experiment. In the same year, Rosalind Franklin and her...The DNA-binding domain can be engineered to target specific DNA sequences while FokI then induces a double...resolved the DNA double helix. In the 1960s, Har Gobind Khorana, Marshall W. Nirenberg, and Robert W. Holley...2018) were highly successful in the box office, indicating the insatiable appetite of the moviegoers for... enzymes generated by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain (FokI). Zinc finger... (TALENs) were engineered. TALENs follow a DNA-binding and DNA-cleaving strategy similar to the ZFNs, ...nucleotide triplets, the components recognize individual nucleotides. This imparts TALENs more specificity...

...oligos that bind with high affinity to small molecules to induce their fluorescence. Until a binding event ...nonspecific binding with RNA by about 7x). Mango fluoresces nearly as brightly as Spinach, but binds with much...small molecule are strongly fluorescent but the binding of an aptamer to its target small molecule activates... process used to evaluate oligonucleotides for binding affinity to a specific target, and the molecular...Broccoli and their derivatives are aptamers able to bind to a small molecule derivative of GFP called 3,5...Additionally, Spinach, the first aptamer identified to bind DFHBI, is highly unstable in vivo due to poor folding...naturally occurring regions in the 5’ UTR of mRNA that bind metabolites, leading to conformational changes that...

... - all without the need to find a llama farm! To find an optimally binding sybody, you want to start out...COVID-19. Researchers identified nanobodies that bind to coronavirus spike proteins (Wrapp et al., 2020...days and isolated special camelid antibodies that bind to the spike proteins in cell culture, and inhibit...small (12-15 kDa) single-domain proteins that can bind effectively to protein targets, similar to how antibodies... “arms” holding the highly variable regions that bind to specific antigens. In contrast, nanobodies are...of sybodies that hypothetically contain ones that bind strongly to your target protein. The Seeger lab ...corresponding to different amino acids) in the regions that bind to the antigen. The Seeger lab can directly provide...

...stringency of your binding. And most importantly: Independently validate identified binding partners with ...the DNA binding domain (DBD) and transcriptional activation (TA) domains retain their individual activities... as using five independent protein interaction detection methods (6). In order to bind DNA and activate...successfully expressed as fusions that retain their individual activities (1). Researchers also discovered the...DBD of the fusion protein shown is still able to bind to the upstream activation sequence (UAS) of its...gene then allows detection of those cells where binding between the proteins of interest has occurred. ...histidine-auxotrophic host strains are used to select for binding-dependent expression of the HIS3 gene, which is...

...modifiers to specific loci without inducing double strand breaks. Below you'll find some of the CRISPR-based epigenetic..., KRAB induced silencing was transient and quickly reversed in culture. However, DNMT3A-induced methylation....2016.08.056 Find plasmids from this publication at Addgene. Maeder ML, Angstman JF, Richardson ME, Linder SJ,...catalytic domain, could mediate demethylation and induce transcription at CpG regions of various promoters...modifiers, and is available as a kit from Addgene.   Find Ready-to-Use Viral Preps for Epigenetic Modification...experience with these constructs in the comments! Find Plasmids for Epigenetic Modification   Leah Schwiesow...Biotechnology 33:510–517 . https://doi.org/10.1038/nbt.3199 Find plasmids from this publication at Addgene. Klann...

...beads and allowed to bind to the antibody being used for IP. These proteins bind only to the Fc portion...Protein G will bind both the primary antibody and serum immunoglobulins indiscriminately, causing competition...triangles. 2) An antibody immobilized on a bead binds specifically to the blue triangle protein but not... During the incubation the immobilized antibody binds to the target protein, thereby tethering it to the...method is desirable if the antibody is expected to bind weakly to the target protein or if the target is... beads in a way that also allows the antibody to bind to the target protein. One of the most common immobilization...cell wall proteins, Protein A and Protein G, which bind to the fragment crystallizable region (Fc) of antibodies...

...transcribed promoters Inducible with galactose; repressible with glucose  Can be used independently or together....not induced. Requires T7 RNA polymerase, which is also controlled by lac operator. Can be induced by IPTG...presence of glucose or by competitive binding of the anti-inducer fucose Weaker. Commonly found in pBAD...the combination of the promoter (RNA polymerase binding site) and operators (response elements). Promoters...The sequence of the promoter region controls the binding of the RNA polymerase and transcription factors...specificity, the eukaryotic RNAP can recognize and bind to specific promoter elements. This means that the...expression mRNA Tetracycline response element promoter Inducible with Tetracyline or its derivatives.  Typically...

...mechanism behind its activity including an extensive RNA scaffold and a domain needed for DNA unwinding. They...expression. Read the paper in Nature Find the CasX plasmids Drug inducible transcriptional activation and ... the paper in Nucleic Acids Research Find the drug inducible Cas9 plasmids E. coli genome-wide inhibition...suppressor genes (TSG). Find the Mouse Metastasis CRISPR-Cpf1 Double Knockout Library Find the Mouse Tumor ...Addgene.org Read our CRISPR guide Find CRISPR plasmids by function Find recently deposited plasmids ... but we hope that this blog series will help you find new CRISPR tools for your research! This time: ...GeneWeld vectors to create knock-ins CasX Drug inducible CRISPR/Cas9 activation E. coli genome-wide inhibition...

...it easier for you to find and use the plasmids you need. Here's what you'll find in this post: Pooled... possible amino acid mutations of the receptor binding domain (RBD) of the viral spike protein.   ...2021. SARS-CoV-2 infects host cells through binding the human angiotensin converting enzyme 2 (ACE2...receptor. The spike protein RBD responsible for binding ACE2 is the target of most anti-SARS-CoV-2 antibodies...RBD mutations which do not interfere with ACE2 binding, but impact recognition by specific anti-SARS-CoV...characterized, and validated by the Gräslund lab. Recent findings suggest that aaRSs are involved in inflammation...fluorescence, and suspension bead assay (Figure 2). After finding no cross-reactivity, antibody candidates underwent...

...Addgene.org Read our CRISPR guide Find CRISPR plasmids by function Find recently deposited plasmids ...dCas9 (Jinek et al., 2012). Like Cas9, dCas9 can bind to a specific DNA sequence via a targeting gRNA.... 2013), or repression of transcription through binding a promoter region to inhibit association of transcriptional...Fujii, 2013). They call this method engineered DNA-binding molecule-mediated chromatin immunoprecipitation...immunoprecipitation (enChIP) because any engineered DNA-binding molecule, such as Zinc-finger or TAL proteins, can be used...genomic DNA that could then be recognized by a DNA-binding molecule (Hoshino and Fujii, 2009). Other forms...study. In addition to the identification of DNA binding proteins, this method can characterize nucleic ...

...antibody binds a target protein in your sample, then a fluorescently labeled secondary antibody binds that...like. Plus, indirect IF amplifies the signal since multiple secondary antibodies will bind to each primary...Figure 1A), you’ll block, add primary antibody to bind the target, wash, add dye-conjugated secondary antibody...there are a few specific considerations to keep in mind. Most importantly, make sure you are using primary...However, the primary-secondary workflow (a.k.a. indirect IF) is more flexible and efficient, since you ...antibodies that have been validated in IF applications (find IHC validated or ICC validated antibodies from Addgene...and validate the antibody yourself. If you can’t find a suitable antibody against your target protein,...

...the mitochondria (binding FRB), allowing for a rapid and inducible inactivation. Find the Robinson plasmids...that can rapidly induction heterodimerization of proteins that contain rapamycin-binding domains, such as...mitochondrial matrix. Gottlieb’s lab has used an inducible MitoTimer reporter using a Tet-on system (pTRE-Tight-MitoTimer...distinct differences of red-to-green fluorescence in individual mitochondria in the networks marks the differences...protein may upregulate an alternative pathway or induce other compensatory mechanisms. How can protein ... as FKBP and FRB. The cell does not appear to mind extraneous proteins bound to its outer mitochondrial...protein with a FRB domain (MitoTRAP). Rapamycin binds your recombinant protein (via FKBP) and then sequesters...

...in Nature Biotechnology Find the base editors   Single base editor that induces C-to-T and A-to-G mutations...sgRNA scaffold with a com aptamer which binds an aptamer binding protein for packaging into a lentivirus-like...Addgene.org Read our CRISPR guide Find CRISPR plasmids by function Find recently deposited plasmids ... but we hope that this blog series will help you find more new CRISPR tools for your research! This time...Expression of this system is tunable via a rhamnose-inducible promoter. While this system was optimized for ...used in. Read the paper in ACS Synthetic Biology Find the CRISPRi toolkit SECURE adenine base editor...editors into one protein. They fused an activation-induced cytidine deaminase (AID) and an engineered tRNA...